Polynucleic acids isolated from a porcine reproductive and respiratory syndrome virus (PRRSV), proteins encoded by the polynucleic acids, vaccines based on the proteins and/or polynucleic acids, a method of protecting a pig from a PRRSV and a method of detecting a PRRSV

ABSTRACT

The present invention provides a purified preparation containing a polynucleic acid encoding at least one polypeptide selected from the group consisting of proteins encoded by one or more open reading frames (ORF&#39;s) of an Iowa strain of porcine reproductive and respiratory syndrome virus (PRRSV), proteins at least 80% but less than 100% homologous with those encoded by one or more of ORF 2, ORF 3, ORF 4 and ORF 5 of an Iowa strain of PRRSV, proteins at least 97% but less than 100% homologous with proteins encoded by one or both of ORF 6 and ORF 7 of an Iowa strain of PRRSV, antigenic regions of such proteins which are at least 5 amino acids in length and which effectively stimulate immunological protection in a porcine host against a subsequent challenge with a PRRSV isolate, and combinations thereof, in which amino acids non-essential for antigenicity may be conservatively substituted. The present invention also concerns a polypeptide encoded by such a polynucleic acid; a vaccine comprising an effective amount of such a polynucleic acid or protein; antibodies which specifically bind to such a polynucleic acid or protein; methods of producing the same; and methods of raising an effective immunological response against a PRRSV, treating a pig infected by a PRRSV, and detecting a PRRSV.

This is a continuation-in-part of application Ser. No. 08/131,625, filedon Oct. 5, 1993, pending, which is a continuation-in-part of applicationSer. No. 07/969,071, filed on Oct. 30, 1992, now abandoned. The entirecontents of application Ser. No. 08/131,625, filed on Oct. 5, 1993, areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns DNA isolated from a porcine reproductiveand respiratory virus (PRRSV), a protein and/or a polypeptide encoded bythe DNA, a vaccine which protects pigs from a PRRSV based on the proteinor DNA, a method of protecting a pig from a PRRSV using the vaccine, amethod of producing the vaccine, a method of treating a pig infected byor exposed to a PRRSV, and a method of detecting a PRRSV.

2. Discussion of the Background

In recent years, North American and European swine herds have beensusceptible to infection by new strains of reproductive and respiratoryviruses (see A.A.S.P., September/October 1991, pp. 7-11; The VeterinaryRecord, Feb. 1, 1992, pp. 87-89; Ibid., Nov. 30, 1991, pp. 495-496;Ibid., Oct. 26, 1991, p. 370; Ibid., Oct. 19, 1991, pp. 367-368; Ibid.,Aug. 3, 1991, pp. 102-103; Ibid., Jul. 6, 1991; Ibid., Jun. 22, 1991, p.578; Ibid., Jun. 15, 1991, p. 574; Ibid., Jun. 8, 1991, p. 536; Ibid.,Jun. 1, 1991, p. 511; Ibid., Mar. 2, 1991, p. 213). Among the first ofthe new strains to be identified was a virus associated with theso-called Mystery Swine Disease (MSD) or “blue-eared syndrome”, nowknown as Swine Infertility and Respiratory Syndrome (SIRS) or PorcineReproductive and Respiratory Syndrome (PRRS).

An MSD consisting of reproductive failure in females and respiratorydisease in nursing and weaned pigs appeared in the midwestern UnitedStates in 1987 (Hill et al., Am. Assoc. Swine Practitioner Newsletter4:47 (1992); Hill et al., Proceedings Mystery Swine Disease CommitteeMeeting, Denver, Colo. 29-31 (1990); Keffaber, Am. Assoc. SwinePractitioner Newsletter 1:1-9 (1989); Loula, Agri-Practice 12:23-34(1991)). Reproductive failure was characterized by abortions, stillbornand weak-born pigs. The respiratory disease in nursing and weaned pigswas characterized by fever, labored breathing and pneumonia. A similardisease appeared in Europe in 1990 (Paton et al., Vet. Rec. 128:617(1991); Wensvoort et al., Veterinary Quarterly 13:121-130 (1991); Blaha,Proc. Am. Assoc. Swine Practitioners, pp. 313-315 (1993)),-and has nowbeen recognized worldwide.

This disease has also been called porcine epidemic abortion andrespiratory syndrome (PEARS), blue abortion disease, blue ear disease(U.K.), abortus blau (Netherlands), seuchenhafter spatabort der schweine(Germany), Heko-Heko disease, and in the U.S., Wabash syndrome, mysterypig disease (MPD), and swine plague (see the references cited above andMeredith, Review of Porcine Reproductive and Respiratory DiseaseSyndrome, Pig Disease Information Centre, Department of VeterinaryMedicine, Madingley Road, Cambridge CB3 OES, U.K. (1992); Wensvoort etal., Vet. Res. 24:117-124 (1993); Paul et al., J. Clin. Vet. Med.11:19-28 (1993)). In Europe, the corresponding virus has been termed“Lelystad virus.”

At an international conference in May, 1992, researchers from around theworld agreed to call this disease Porcine Reproductive and RespiratorySyndrome (PRRS). The disease originally appeared to be mainly areproductive disease during its early phases, but has now evolvedprimarily into a respiratory disease.

Porcine reproductive and respiratory syndrome virus (PRRSV) is arelatively recently recognized swine pathogen associated with porcinereproductive and respiratory syndrome (PRRS). PRRSV is a significantpathogen in the swine industry. PRRSV infections are common in the U.S.swine herds. Outbreaks of PRRS in England have led to cancellation ofpig shows.

The symptoms of PRRS include a reluctance to eat (anorexia), a mildfever (pyrexia), cyanosis of the extremities (notably bluish ears),stillbirths, abortion, high mortality in affected litters, weak-bornpiglets and premature farrowing. The majority of piglets born alive toaffected sows die within 48 hours. PRRS clinical signs include mildinfluenza-like signs, rapid respiration (“thumping”), and a diffuseinterstitial pneumonitis. PRRS virus has an incubation period of about1-2 weeks from contact with a PRRSV-infected animal. The virus appearsto be an enveloped RNA arterivirus (The Veterinary Record, Feb. 1,1992). The virus has been grown successfully in pig alveolar macrophagesand CL2621 cells (Benfield et al, J. Vet. Diagn. Invest., 4:127-133,1992; Collins et al, Swine Infertility and Respiratory Syndrome/MysterySwine Disease. Proc., Minnesota Swine Conference for Veterinarians, pp.200-205, 1991), and in MARC-145 cells (Joo, PRRS: Diagnosis, Proc.,Allen D. Leman Swine Conference, Veterinary Continuing Education andExtension, University of Minnesota (1993), 20:53-55; Kim et al, Arch.Virol., 133:477-483 (1993)). A successful culturing of a virus whichcauses SIRS has also been reported by Wensvoort et al (Mystery SwineDisease in the Netherlands: The Isolation of Lelystad Virus. Vet. Quart.13:121-130, 1991).

Initially, a number of agents were incriminated in the etiology of thisdisease (Wensvoort et al., Vet. Res. 24:117-124 (1993); Woolen et al.,J. Am. Vet. Med. Assoc. 197:600-601 (1990)). There is now a consensusthat the causative agent of PRRS is an enveloped RNA virus referred toas Porcine Reproductive and Respiratory Syndrome Virus (PRRSV),reportedly of approximately 62 nm in diameter (Benfield et al., J. Vet.Diagn. Invest., 4:127-133, 1992).

Virus isolates vary in their ability to replicate in continuous celllines. Some grow readily, while others require several passages and somegrow only in swine alveolar (SAM) cultures (Bautista et al., J. Vet.Diagn. Invest. 5:163-165, 1993; see also the Examples hereunder[particularly Table 1]).

PRRSV is a member of an Arterivirus group which includes equinearteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) andsimian hemorrhagic fever virus (SHFV) (Benfield et al., 1992, supra;Plagemann, Proc. Am. Assoc. Swine Practitioners, 4:8-15 1992; Plagemannand Moennig, Adv. Virus Res. 41:99-192, 1992; Conzelmann et al.,Virology, 193:329-339, 1993; Godney et al., Virology, 194:585-596, 1993;Meulenberg et al., Virology, 192:62-72, 1993). The positive-strand RNAviruses of this Arterivirus group resemble togaviruses morphologically,but are distantly related to coronaviruses and toroviruses on the basisof genome organization and gene expression (Plagemann et al., supra;Spaan et al., J. Gen. Virol. 69, 2939-2952 (1988); Strauss et al., Annu.Rev. Biochem. 42, 657-683 (1988); Lai, Annu. Rev. Microbiol. 44, 303-333(1990); Snijder et al., Nucleic Acid Res. 18, 4535-4542 (1990)). Themembers of this group infect macrophages and contain a nested set of 5to 7 subgenomic mRNAs in infected cells (Plagemann et al., supra;Meulenberg et al., Virology, 192, 62-72 (1993); Conzelmann et al.,Virology, 193, 329-339 (1993); 15, 16, 17, 18, 19).

The viral genome of European isolates has been shown to be a plusstranded RNA of about 15.1 kb (Conzelmann et al., supra; Meulenberg etal., supra), and appears to be similar in genomic organization to LDVand EAV (Meulenberg et al., supra). However, no serologicalcross-reaction has been found among PRRSV, LDV and EAV (Goyal et al., J.Vet. Diagn. Invest., 5, 656-664 (1993)).

PRRSV was initially cultivated in swine alveolar macrophage (SAM) cellcultures (Pol et al., Veterinary Quarterly, 13:137-143, 1991; Wensvoortet al., Veterinary Quarterly, 13:121-130, 1991) and then in continuouscell lines CL2621 (Benfield et al., supra), MA-104, and MARC-145 (Joo,Proc. Allen D. Leman Swine Conference, pp. 53-55, 1993). Thereproductive and respiratory disease has been reproduced with cell freelung filtrates (Christianson et al., Am. J. Vet. Res., 53:485-488, 1992;Collins et al., J. Vet. Diagn. Invest., 4:117-126, 1992; Halbur etal.,Proc. Central Veterinary Conference, pp. 50-59, 1993), and with cellculture-propagated PRRSV (Collins et al., supra, and Proc. Allen D.Leman Swine Conference, pp. 47-48, 1993).

Eight open reading frames (also referred to herein as “ORFs” or “genes”)have been identified in a European PRRSV isolate. The genes of thisEuropean isolate are organized similarly to that in coronavirus(Meulenberg et al., supra). A 3′-end nested set of messenger RNA hasbeen found in PRRSV-infected cells similar to that in coronaviruses(Conzelmann et al., supra; Meulenberg et al., supra).

The ORF 1a and 1b at the 5′-half of the European PRRSV genome arepredicted to encode viral RNA polymerase. The ORF's 2-6 at the 3′-halfof the genome likely encode for viral membrane-associated (envelope)proteins (Meulenberg et al., supra). ORF6 is predicted to encode themembrane protein (M) based on its similar characteristics with the ORF 6of EAV, ORF 2 of LDV, and the M protein of mouse hepatitis virus andinfectious bronchitis virus (Meulenberg et al., Virology 192, 62-72(1993); Conzelmann et al., Virology 193, 329-339 (1993); Murtaugh, Proc.Allen D. Leman Swine Conference, Minneapolis, Minn., pp. 43-45 (1993);Mardassi et al., Abstracts of Conference of Research Workers in AnimalDiseases, Chicago, Ill., pp. 43 (1993)). The product of ORF 7 isextremely basic and hydrophilic, and is predicted to be the viralnucleocapsid protein (N) (Meulenberg et al., supra; Conzelmann et al.,supra; Murtaugh, supra; Mardassi et al., supra and J. Gen. Virol.,75:681-685 (1994)).

Although conserved epitopes have been identified between U.S. andEuropean PRRSV isolates using monoclonal antibodies (Nelson et al., J.Clin. Microbiol., 31:3184-3189, 1993), there is extensive antigenic andgenetic variation both among U.S. and European isolates of PRRSV(Wensvoort et al., J. Vet. Diagn. Invest., 4:134-138, 1992). Europeanisolates are genetically closely related, as the nucleotide sequence atthe 3′-half of the genome from two European PRRSV isolates is almostidentical (Conzelmann et al., supra; Meulenberg et al., supra).

Although the syndrome caused by PRRSV appears to be similar in the U.S.and Europe, several recent studies have described phenotypic, antigenic,genetic and pathogenic variations among PRRSV isolates in the U.S. andin Europe (Murtaugh, supra; Bautista et al., J. Vet. Diagn. Invest., 5,163-165 (1993); Bautista et al., J. Vet. Diagn. Invest., 5, 612-614(1993); Wensvoort et al., J. Vet. Diagn. Invest., 4, 134-138 (1992);Stevenson et al., J. Vet. Diagn. Invest., 5, 432-434 (1993)). Forexample, the European isolates grow preferentially in SAM cultures andreplicate to a very low titer in other culture systems (Wensvoort, Vet.Res., 24, 117-124 (1993); Wensvoort et al., J. Vet. Quart., 13, 121-130(1991); Wensvoort et al., J. Vet. Diagn. Invest., 4, 134-138 (1992)). Onthe other hand, some of the U.S. isolates have been shown to replicatewell in SAM as well as in the continuous cell line CL2621 (Benfield etal., J. Vet. Diagn. Invest., 4, 127-133 (1992); Collins et al., J. Vet.Diagn. Invest., 4, 117-126 (1992)). Thus, phenotypic differences amongU.S. isolates are observed, as not all PRRSV isolates isolated on SAMcan replicate on the CL2621 cell line (Bautista et al., J. Vet. Diagn.Invest., 5, 163-165 (1993)).

A high degree of regional antigenic variation among PRRSV isolates mayexist. Four European isolates were found to be closely relatedantigenically, but these European isolates differed antigenically fromU.S. isolates. Further, three U.S. isolates were shown to differantigenically from each other (Wensvoort et al., J. Vet. Diagn. Invest.,4, 134-138 (1992)). Animals seropositive for European isolates werefound to be negative for U.S. isolate VR 2332 (Bautista et al., J. Vet.Diagn. Invest., 5, 612-614 (1993)).

U.S. PRRSV isolates differ genetically at least in part from Europeanisolates (Conzelmann et al., supra; Meulenberg et al., supra; Murtaughet al., Proc. Allen D. Leman Conference, pp. 43-45, 1993). The geneticdifferences between U.S. and European isolates are striking, especiallysince they are considered to be the same virus (Murtaugh, supra).Similar observations were also reported when comparing the Canadianisolate IAF-exp91 and another U.S. isolate VR 2332 with LV (Murtaugh,supra; Mardassi, supra). However, the 3′ terminal 5 kb nucleotidesequences of two European isolates are almost identical (Conzelmann etal., supra; Meulenberg et al., supra).

The existence of apathogenic or low-pathogenic strains among isolateshas also been suggested (Stevenson, supra). Thus, these studies suggestthat the PRRSV isolates in North America and in Europe are antigenicallyand genetically heterogeneous, and that different genotypes or serotypesof PRRSV exist. However, prior to the present invention, the role ofantigenic and genetic variation in the pathogenesis of PRRSV was notentirely clear.

The occurrence of PRRS in the U.S. has adversely affected the pigfarming industry. Almost half of swine herds in swine-producing statesin the U.S. are seropositive for PRRSV (Animal Pharm., 264:11 (Nov. 11,1992)). In Canada, PRRS has been characterized by anorexia and pyrexiain sows lasting up to 2 weeks, late-term abortions, increased stillbirthrates, weak-born pigs and neonatal deaths preceded by rapid abdominalbreathing and diarrhea. Work on the isolation of the virus causing PRRS,on a method of diagnosing PRRS infection, and on the development of avaccine-against the PRRS virus has been published (see Canadian PatentPublication No. 2,076,744; PCT International Patent Publication No. WO93/03760; PCT International Patent Publication No. WO 93/06211; and PCTInternational Patent Publication No. WO 93/07898).

There is also variability in the virulence of PRRSV in herds. Recently,a more virulent form of PRRS has been occurring with increased incidencein 3-8 week old pigs in the midwestern United States. Typically, healthy3-5 week old pigs are weaned and become sick 5-7 days later. Routinevirus identification methods on tissues from affected pigs have shownthat swine influenza virus (SIV), pseudorabies virus (PRV), andMycoplasma hyopneumoniae are not associated with this new form of PRRS.Originally termed proliferative interstitial pneumonia (PIP; see U.S.patent application Ser. No. 07/969,071), this disease has been veryrecently linked with PRRS, and the virus has been informally named the“Iowa strain” of PRRSV (see U.S. patent application Ser. No.08/131,625).

Pessimism and skepticism has been expressed in the art concerning thedevelopment of effective vaccines against these porcine viruses (TheVeterinary Record, Oct. 26, 1991). A belief that human influenza vaccinemay afford some protection against the effects of PRRS and PNP exists(The Veterinary Record, Jul. 6, 1991).

Viral envelope proteins are known to be highly variable in manycoronaviruses, such as feline infectious peritonitis virus and mousehepatitis virus (Dalziel et al: Site-specific alteration of murinehepatitis virus type 4 peplomer glycoprotein E2 results in reducedneurovirulence. J. Virol., 59:464-471 (1986); Fleming et al:Pathogenicity of antigenic variants of murine coronavirus JHM selectedwith monoclonal antibodies. J. Virol., 58:869-875 (1986); Fiscus et al:Antigenic comparison of the feline coronavirus isolates; Evidence formarkedly different peplomer glycoproteins. J. Virol., 61:2607-2613(1987); Parker et al: Sequence analysis reveals extensive polymorphismand evidence of deletions within the E2 glycoprotein gene of severalstrains of murine hepatitis virus. Virology, 173:664-673 (1989)).

For example, a deletion or a mutation in the major envelope protein incoronaviruses can alter tissue tropism and in vivo pathogenicity. Amutation in a monoclonal antibody-resistant mutant of MHV has resultedin loss of its neurovirulence for mice (Fleming et al, 1986 supra).Porcine respiratory coronavirus (PRCV) is believed to be a deletionmutant of transmissible gastroenteritis virus (TGEV) in swine. Thedeletion in the PRCV genome may be in the 5′-end of the spike (S) geneof TGEV (Halbur et al, An overview of porcine viral respiratory disease.Proc. Central Veterinary Conference, pp. 50-59 (1993); Laude et al,Porcine respiratory coronavirus: Molecular features and virus-hostinteractions. Vet. Res., 24:125-150 (1993); Vaughn et al, Isolation andcharacterization of three porcine respiratory coronavirus isolates withvarying sizes of deletions. J. Clin. Micro., 32:1809-1812 (1994)).

PRCV has a selective tropism for the respiratory tract and does notreplicate in the gastrointestinal tract (Rasschaert et al, Porcinerespiratory coronavirus differs from transmissible gastroenteritis virusby a few genomic deletions. J. Gen. Virol., 71:2599-2607 (1990); Laudeet al, 1993 supra). In contrast, TGEV has a tropism for both respiratoryand gastrointestinal tracts (Laude et al, 1993 supra).

Variation in antigenic and genetic relatedness among LDV isolates ofvarying pathogenicity is also known (Kuo et al,Lactate-dehydrogenase-elevating virus (LDV): subgenomic mRNAs, mRNAleader and comparison of 3′-terminal sequences of two LDV isolates.Virus Res., 23:55-72 (1992); Plagemann, LDV, EAV, and SHFV: A new groupof positive stranded RNA viruses. Proc. Am. Assoc. Swine Practitioners,4:8-15 (1992); Chen et al, Sequences of 3′ end of genome and of 5′ endof open reading frame 1a of lactate dehydrogenase-elevating virus andcommon junction motifs between 5′ leader and bodies of seven subgenomicmRNAs. J. Gen. Virol., 74:643-660 (1993)).

However, the present invention provides the first insight into therelationships between the open reading frames of the PRRSV genome andtheir corresponding effects on virulence and replication.

Further, a diagnosis of porcine reproductive and respiratory syndrome(PRRS) relies on compiling information from the clinical history of theherd, serology, pathology, and ultimately on isolation of the PRRS virus(PRRSV). Three excellent references reviewing diagnosis of PRRSV havebeen published in the last year (Van Alstine et al, “Diagnosis ofporcine reproductive and respiratory syndrome,” Swine Health andProduction, Vol. 1, No. 4 (1993), p. 24-28; Christianson et al, “Porcinereproductive and respiratory syndrome: A review.” Swine Health andProduction, Vol. 1, No. 2 (1994), pp. 10-28 and Goval, “Porcinereproductive and respiratory syndrome,” J. Vet. Diagn. Invest. 5:656-664(1993)). PRRSV has also recently been shown to replicate in pulmonaryalveolar macrophages by gold colloid immunohistochemistry (Magar et al(1993): Immunohistochemical detection of porcine reproductive andrespiratory syndrome virus using colloidal gold. Can. J. Vet. Res.,57:300-304).

Clinical signs vary widely between farms, and thus, are not the mostreliable evidence of a definitive diagnosis, except in the case of asevere acute outbreak in naive herds which experience abortion storms,increased numbers of stillborn pigs, and severe neonatal and nursery pigpneumonia. Presently, the most common clinical presentation is pneumoniaand miscellaneous bacterial problems in 3-10 week old pigs. However,many PRRSV-positive herds have no apparent reproductive or respiratoryproblems.

Some herds evidence devastating reproductive failure, characterized bythird-trimester abortions, stillborn pigs and weak-born pigs. Many ofthese herds also experience severe neonatal respiratory disease.Respiratory disease induced by PRRSV in 4-10 week-old pigs is alsocommon and can be quite severe (Halbur et al, Viral contributions to theporcine respiratory disease complex. Proc. Am. Assoc. Swine Pract.(1993), pp. 343-350). Clinical PRRSV outbreaks are frequently followedby bacterial pneumonia, septicemia, or enteritis. Thus, it has beendifficult to obtain an acceptably rapid and reliable diagnosis ofinfection by PRRSV, prior to the present invention.

The pig farming industry has been and will continue to be adverselyaffected by these porcine reproductive and respiratory diseases and newvariants thereof, as they appear. PRRSV is a pathogen of swine thatcauses economic losses from reproductive and respiratory diseases.Economic losses from PRRS occur from loss of pigs from abortions,stillborn pigs, repeat breeding, pre-weaning and postweaning mortality,reduced feed conversion efficiency, increased drug and labor cost andhave been estimated to cost approximately $236 per sow in addition toloss of profits (Polson et al., Financial implications of mystery swinedisease (MSD), Proc. Mystery Swine Disease Committee Meeting, Denver,Colo., 1990, pp. 8-28). This represents a loss of $23,600 for a 100 sowherd or $236,000 for a 1000 sow herd.

PRRSV causes additional losses from pneumonia in nursery pigs. However,the exact economic losses from PRRSV-associated pneumonia are not known.PRRSV is an important cause of pneumonia in nursery and weaned pigs.Reproductive disease was the predominant clinical outcome of PRRSVinfections during the past few years. Respiratory disease has now becomethe main problem associated with PRRSV.

Surprisingly, the market for animal vaccines in the U.S. and worldwideis larger than the market for human vaccines. Thus, there exists aneconomic incentive to develop new veterinary vaccines, in addition tothe substantial public health benefit which is derived from protectingfarm animals from disease.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide apolynucleic acid isolated from a porcine reproductive and respiratoryvirus (PRRSV).

It is a further object of the present invention to provide an isolatedpolynucleic acid which encodes a PRRSV protein.

It is a further object of the present invention to provide a PRRSVprotein, either isolated from a PRRSV or encoded by a PRRSV polynucleicacid.

It is a further object of the present invention to provide a protein- orpolynucleic acid-based vaccine which protects a pig against PRRS.

It is a further object of the present invention to provide a method ofraising an effective immunological response against a PRRSV using thevaccine.

It is a further object of the present invention to provide a method ofproducing a protein- or polynucleic acid-based vaccine which protects apig against a PRRSV infection.

It is a further object of the present invention to provide a method oftreating a pig infected by or-exposed to a PRRSV.

It is a further object of the present invention to provide a method ofdetecting PRRSV.

It is a further object of the present invention to provide animmunoperoxidase diagnostic assay for detection of PRRSV antigen inporcine tissues.

It is a further object of the present invention to provide an antibodywhich immunologically binds to a PRRSV protein or to an antigenic regionof such a protein.

It is a further object of the present invention to provide an antibodywhich immunologically binds to a protein- or polynucleic acid-basedvaccine which protects a pig against a PRRSV.

It is a further object of the present invention to provide a method oftreating a pig exposed to or infected by a PRRSV.

It is a further object of the present invention to provide a method ofdetecting and a diagnostic kit for assaying a PRRSV.

It is a further object of the present invention to provide the aboveobjects, where the PRRS virus is the Iowa strain of PRRSV.

These and other objects which will become apparent during the followingdescription of the preferred embodiments, have been provided by at leastone purified polypeptide selected from the group consisting of proteinsencoded by one or more open reading frames (ORF's) of an Iowa strain ofporcine reproductive and respiratory virus (PRRSV), proteins at least80% but less than 100% homologous with those encoded by one or more ofORF 2, ORF 3, ORF 4 and ORF 5 of an Iowa strain of PRRSV, proteins atleast 97% but less than 100% homologous with proteins encoded by one orboth of ORF 6 and ORF 7.of an Iowa strain of PRRSV, antigenic regions ofsaid proteins which are at least 5 amino acids in length and whicheffectively stimulate immunological protection in a porcine host againsta subsequent challenge with a PRRSV isolate, and combinations thereof;an isolated polynucleic acid which encodes such a polypeptide orpolypeptides; a vaccine comprising an effective amount of such apolynucleotide or polypeptide(s); antibodies which specifically bind tosuch a polynucleotide or polypeptide; methods of producing the same; andmethods of raising an effective immunological response against a PRRSV,treating a pig exposed to or infected by a PRRSV, and detecting a PRRSVusing the same.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flowchart outlining a procedure for producing a subunitvaccine;

FIG. 2 is a flowchart outlining a procedure for producing a geneticallyengineered vaccine;

FIG. 3 shows a general schematic procedure for the construction of acDNA λ library as described by the manufacturer (Stratagene);

FIG. 4 shows a general schematic procedure for identifying authenticclones of the PRRS virus isolate ISU-12 (VR 2385) by differentialhybridization (modified from “Recombinant DNA,” 2nd ed., Watson, J. D.,et al., eds. (1992), p. 110);

FIG. 5 is a Northern blot showing the VR 2385 subgenomic mRNA species,denatured with 6 M glyoxal and DMSO, and separated on a 1.5% agarosegel;

FIG. 6 shows the λ cDNA clones used to obtain the 3′-terminal nucleotidesequence of VR 2385;

FIG. 7 shows the 2062-bp 3′-terminal sequence (SEQ ID NO:13) and theamino acid sequences encoded by ORF's 5, 6 and 7 (SEQ ID NOS:15, 17 and19, respectively) of VR 2385;

FIG. 8 compares the ORF-5 regions of the genomes of VR 2385 and Lelystadvirus;

FIG. 9 compares the ORF-6 regions of the genomes of VR 2385 and Lelystadvirus;

FIG. 10 compares the ORF-7 regions of the genomes of VR 2385 andLelystad virus;

FIG. 11 compares the 3′-nontranslational regions of the genomes of VR2385 and Lelystad virus;

FIG. 12 shows a cytopathic effect in HI-FIVE cells infected with arecombinant baculovirus containing the VR 2385 ORF-7 gene(Baculo.PRRSV.7);

FIG. 13 shows HI-FIVE cells infected with a recombinant baculoviruscontaining the VR 2385 ORF-6 gene, stained with swine antisera to VR2385, followed by fluorescein-conjugated anti-swine IgG;

FIG. 14 shows HI-FIVE cells infected with a recombinant baculoviruscontaining the VR 2385 ORF-7 gene, respectively, stained with swineantisera to VR 2385, followed by fluorescein-conjugated anti-swine IgG;

FIG. 15 shows a band of expected size for the VR 2385 ORF-6 product,detected by a radioimmunoprecipitation technique (see Experiment II(B)below);

FIG. 16 shows a band of expected size for the VR 2385 ORF-7 product,detected by a radioimmunoprecipitation technique (see Experiment II(B)below);

FIG. 17 compares the ORF 6 and ORF 7 nucleotide sequences of six U.S.PRRSV isolates and of LV, in which the VR 2385 nucleotide sequence isshown first, and in subsequent sequences, only those nucleotides whichare different are indicated;

FIGS. 18(A)-(B) show the alignment of amino acid sequences of theputative M (FIG. 18(A)) and N (FIG. 18(B)) genes of the proposedarterivirus group, performed with a GENEWORKS program (IntelliGenetics,Inc.);

FIGS. 19(A)-(B) show phylogenetic trees based on the amino acidsequences of the putative M (FIG. 19(A)) and N genes (FIG. 19(B)) forthe proposed arterivirus group;

FIG. 20 shows the nucleotide sequence of a region of the genome of PRRSVisolate VR 2385 containing ORF's 2, 3 and 4;

FIGS. 21(A)-(C) compare the nucleotide sequences of ORF 2, ORF 3 and ORF4 of PRRSV-VR 2385 with the corresponding ORF's of Lelystad virus (LV);

FIGS. 22(A)-(C) show alignments of the predicted amino acid sequencesencoded by ORF's 2, 3 and 4 of PRRSV VR 2385 and LV;

FIG. 23 shows an immunohistochemical stain of a lung tissue sample takenfrom a pig infected 9 days previously with PRRSV, in which positive ABCstaining with hematoxylin counterstain is observed within the cytoplasmof macrophages and sloughed cells in the alveolar spaces;

FIG. 24 shows an immunohistochemical stain of a lung tissue sample takenfrom a pig infected 4 days previously with PRRSV, in which positive ABCstaining with hematoxylin counterstain is demonstrated within cellulardebris in terminal airway lumina;

FIG. 25 shows a heart from a pig infected 9 days previously with PRRSV,in which positive staining is demonstrated within endothelial cells(arrow) and isolated macrophages by the present streptavidin-biotincomplex method (with hematoxylin counterstain); the bar indicates alength of 21 microns;

FIG. 26 shows a tonsil from a pig infected 9 days previously with PRRSV,in which positive staining cells (arrow heads) are demonstrated withinfollicles and in the crypt epithelium by the present streptavidin-biotincomplex method (with hematoxylin counterstain); the bar indicates alength of 86 microns;

FIG. 27 shows a lymph node from a pig infected 9 days previously withPRRSV, in which positive staining is demonstrated within follicles bythe present streptavidin-biotin complex method (with hematoxylincounterstain), and positive cells (arrows) resemble macrophages ordendritic cells; the bar indicates a length of 21 microns;

FIGS. 28(A)-(C) are photomicrographs of lungs from pig inoculated with(A) culture fluid from an uninfected cell line, (B) culture fluid from acell line infected with a low virulence PRRSV isolate (the lungs showPRRS-A type lesions), and (C) culture fluid from a cell line infectedwith a high virulence PRRSV isolate (the lungs show PRRS-B type lesions)

FIGS. 29(A)-(B) illustrate immunohistochemical staining with anti-PRRSVmonoclonal antibody of a lung from a pig infected 9 days previously withPRRSV; and

FIGS. 30(A)-(B) show Northern blots of PRRSV isolates VR 2385pp(designated as “12”), VR 2429 (ISU-22, designated as “22”), VR 2430,designated as “55”), ISU-79 (designated as “79”), ISU-1894 (designatedas “1894”), and VR 2431, designated as “3927”).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the present invention, a “porcine reproductive and respiratorysyndrome virus” or “PRRSV” refers to a virus which causes the diseasesPRRS, PEARS, SIRS, MSD and/or PIP (the term “PIP” now appears to bedisfavored), including the Iowa strain of PRRSV, other strains of PRRSVfound in the United States (e.g., VR 2332), strains of PRRSV found inCanada (e.g., IAF-exp91), strains of PRRSV found in Europe (e.g.,Lelystad virus, PRRSV-10), and closely-related variants of these viruseswhich may have appeared and which will appear in the future.

The present vaccine is effective if it protects a pig against infectionby a porcine reproductive and respiratory syndrome virus (PRRSV). Avaccine protects a pig against infection by a PRRSV if, afteradministration of the vaccine to one or more unaffected pigs, asubsequent challenge with a biologically pure virus isolate (e.g., VR2385, VR 2386, or other virus isolate described below) results in alessened severity of any gross or histopathological changes (e.g.,lesions in the lung) and/or of symptoms of the disease, as compared tothose changes or symptoms typically caused by the isolate in similarpigs which are unprotected (i.e., relative to an appropriate control).More particularly, the present vaccine may be shown to be effective byadministering the vaccine to one or more suitable pigs in need thereof,then after an appropriate length of time (e.g., 1-4 weeks), challengingwith a large sample (10³⁻⁷ TCID₅₀) of a biologically pure PRRSV isolate.A blood sample is then drawn from the challenged pig after about oneweek, and an attempt to isolate the virus from the blood sample is thenperformed (e.g., see the virus isolation procedure exemplified inExperiment VIII below). Isolation of the virus is an indication that thevaccine may not be effective, and failure to isolate the virus is anindication that the vaccine may be effective.

Thus, the effectiveness of the present vaccine may be evaluatedquantitatively (i.e., a decrease in the percentage of consolidated lungtissue as compared to an appropriate control group) or qualitatively(e.g., isolation of PRRSV from blood, detection of PRRSV antigen in alung, tonsil or lymph node tissue sample by an immunoperoxidase assaymethod (described below], etc.). The symptoms of the porcinereproductive and respiratory disease may be evaluated quantitatively(e.g., temperature/ fever), semi-quantitatively (e.g., severity ofrespiratory distress [explained in detail below], or qualitatively(e.g., the presence or absence of one or more symptoms or a reduction inseverity of one or more symptoms, such as cyanosis, pneumonia, heartand/or brain lesions, etc.).

An unaffected pig is a pig which has either not been exposed to aporcine reproductive and respiratory disease infectious agent, or whichhas been exposed to a porcine reproductive and respiratory diseaseinfectious agent but is not showing symptoms of the disease. An affectedpig is one which shows symptoms of PRRS or from which PRRSV can beisolated.

The clinical signs or symptoms of PRRS may include lethargy, respiratorydistress, “thumping” (forced expiration), fevers, roughened haircoats,sneezing, coughing, eye edema and occasionally conjunctivitis. Lesionsmay include gross and/or microscopic lung lesions, myocarditis,lymphadenitis, encephalitis and rhinitis. The infectious agent may be asingle virus, or may be combined with one or more additional infectiousagents (e.g., other viruses or bacteria). In addition, less virulent andnon-virulent forms of the PRRSV and of Iowa strain have been found,which may cause either a subset of the above symptoms or no symptoms atall. Less virulent and non-virulent forms of PRRSV can be used accordingto the present invention to provide protection against porcinereproductive and respiratory diseases nonetheless.

Histological lesions in the various porcine diseases are different.Table I below compares physiological observations and pathology of thelesions associated with a number of diseases caused by porcine viruses:TABLE I Swine Viral Pneumonia Comparative Pathology Lesion PRRS(p)PRRS(o) SIV PNP PRCV PPMV Iowa Type II + +++ + +++ ++ ++ ++++ Inter.thickening ++++ + + + ++ ++ + Alveolar exudate + +++ ++ ++ ++ ++ +++Airway necrosis − − ++++ ++++ +++ + − Syncytia − ++ +/− ++ + + +++Encephalitis + +++ − − − ++ + Myocarditis +/− ++ − − − − +++wherein “PRRS(p)” represents the published pathology of the PRRS virus,“PRRS(o)” represents the pathology of PRRS virus observed by the presentInventors, “SIV” represents swine# influenza A virus, “PRCV” represents porcine respiratory coronavirus,“PPMV” represents porcine paramyxovirus, “Iowa” refers to the strain ofPRRSV discovered by the present # Inventors, “Type II” refers to Type IIpneumocytes (which proliferate in infected pigs), “Inter.” refers tointerstitial septal infiltration by mononuclear cells, “Airway necrosis”refers to necrosis in terminal # airways, and the symbols (−) and (+)through (++++) refer to a comparative severity scale as follows:(−): negative (not observed)(+): mild (just above the threshold of observation)(++): moderate(+++): severe(++++): most severe

A “porcine reproductive and respiratory virus” or “PRRSV” causes aporcine reproductive and respiratory disease defined by one or more ofthe clinical signs, symptoms, lesions and histopathology as describedabove, and is characterized as being an enveloped RNA arterivirus,having a size of from 50 to 80 nm in diameter and from 250 to 400 nm inlength. “North American strains of PRRSV” refer to those strains ofPRRSV which are native to North America. “U.S. strains of PRRSV” referto strains of PRRSV native to the U.S., and “European strains of PRRSV”refer to strains native to Europe, such as Lelystad virus (deposited bythe CDI [Lelystad, Netherlands] in the depository at the InstitutPasteur, Paris, France, under the deposit number I-1102; seeInternational Patent Publication No. WO 92/21375, published on Dec. 10,1992).

The “Iowa strain” of PRRSV refers to (a) those strains of PRRSV isolatedby the presented Inventors, (b) those strains having at least a 970sequence identity (or homology) in the seventh open reading frame (ORF7) with at least one of VR 2385, VR 2430 and VR 2431; (c) strains which,after no more than 5 passages, grow to a titer of at least 10⁴ TCIDS₅₀in CRL 11171 cells, MA-104 cells or PSP-36 cells, (d) those strainshaving at least 80% and preferably at least 90% homology with one ormore of ORF's 2-5 of VR 2385, and (e) those strains which cause agreater percentage consolidation of lung tissue than Lelystad virus(e.g., at 10 days post-infection, infected pigs exhibit at least 20% andpreferably at least 40% lung consolidation). Preferably, the Iowa strainof PRRSV is characterized by at least two of the above characteristics(a)-(e).

The present invention is primarily concerned with polynucleic acids(segments of genomic RNA and/or DNA, mRNA, cDNA, etc.) isolated from orcorresponding to a porcine reproductive and respiratory syndrome virus(PRRSV), proteins encoded by the DNA, methods of producing thepolynucleic acids and proteins, vaccines which protect pigs from aPRRSV, a method of protecting a pig from a PRRSV using the vaccine, amethod of producing the vaccine, a method of treating a pig infected byor exposed to a PRRSV, and a method of detecting a PRRSV. Moreparticularly, the present invention is concerned with a vaccine whichprotects pigs from North American strains of PRRSV, a method ofproducing and administering the vaccine, and polynucleic acids andproteins obtained from an Iowa strain of PRRSV. However, it is believedthat the information learned in the course of developing the presentinvention will be useful in developing vaccines and methods ofprotecting pigs against any and/or all strains of porcine reproductiveand respiratory syndrome. Therefore, the present invention is notnecessarily limited to polynucleic acids, proteins, vaccines and methodsrelated to the Iowa strain of PRRS virus (PRRSV).

The phrase “polynucleic acid” refers to RNA or DNA, as well as mRNA andcDNA corresponding to or complementary to the RNA or DNA isolated fromthe virus or infectious agent. An “ORF” refers to an open reading frame,or polypeptide-encoding segment, isolated from a viral genome, includingthe PRRSV genome. In the present polynucleic acid, an ORF can beincluded in part (as a fragment) or in whole, and can overlap with the5′- or 3′-sequence of an adjacent ORF (see FIGS. 7 and 21, andExperiments I and IV below). A “polynucleotide” is equivalent to apolynucleic acid, but may define a distinct molecule or group ofmolecules (e.g., as a subset of a group of polynucleic acids).

Referring now to FIGS. 1-2, flowcharts of procedures are provided forpreparing types of vaccines encompassed by the present invention. Theflowcharts of FIGS. 1-2 are provided as exemplary methods of producingthe present vaccines, and are not intended to limit the presentinvention in any manner.

The first step in each procedure detailed in FIGS. 1-2 is to identify acell line susceptible to infection with a porcine reproductive andrespiratory virus or infectious-agent. (To simplify the discussionconcerning preparation of the vaccine, the term “virus” refers to avirus and/or other infectious agent associated with a porcinereproductive and respiratory disease.) A master cell stock (MCS) of thesusceptible host cell is then prepared. The susceptible host cellscontinue to be passaged beyond MCS. Working cell stock (WCS) is preparedfrom cell passages between MCS and MCS+n.

A master seed virus is propagated on the susceptible host cell line,between MCS and MCS+n, preferably on WCS. The raw virus is isolated bymethods known in the art from appropriate, preferably homogenized,tissue samples taken from infected pigs exhibiting disease symptomscorresponding to those caused by the virus of interest. A suitable hostcell, preferably a sample of the WCS, is infected with the raw virus,then cultured. Vaccine virus is subsequently isolated andplaque-purified from the infected, cultured host cell by methods knownin the art. Preferably, the virus to be used to prepare the vaccine isplaque-purified three times;

Master seed virus (MSV) is then prepared from the plaque-purified virusby methods known in the art. The MSV(X) is then passaged in WCS at leastfour times through MSV(X+1), MSV(X+2), MSV(X+3) and MSV(X+4) viruspassages. The MSV(X+4) is considered to be the working seed virus.Preferably, the virus passage to be used in the pig studies and vaccineproduct of the present invention is MSV(X+5), the product of the fifthpassage.

In conjunction with the working cell stock, the working seed virus iscultured by known methods in sufficient amounts to prepare a prototypevaccine, preferably MSV(X+5). The present prototype vaccines may be ofany type suitable for use in the veterinary medicine field. The primarytypes of vaccines on which the present invention focuses include asubunit vaccine (FIG. 1) and a genetically engineered vaccine (FIG. 2).However, other types of vaccines recognized in the field of veterinaryvaccines, including live, modified live, attenuated and killed virusvaccines, are also acceptable. A killed vaccine may be rendered inactivethrough chemical treatment or heat, etc., in a manner known to theartisan of ordinary skill.

An attenuated virus may be obtained by repeating serial passage of thevirus in a suitable host cell a sufficient number of times to obtain anessentially non-virulent virus. For example, a PRRSV may be seriallypassaged from 1 to 20 times (or more, if desired), in order to render itsufficiently attenuated for use as an attenuated vaccine. MSV(X+5) maybe such an attenuated vaccine.

In the procedures outlined by each of FIGS. 1-2, following preparationof a prototype vaccine, pig challenge models and clinical assays areconducted by methods known in the art. For example, before performingactual vaccination/challenge studies, the disease to be prevented and/ortreated must be defined in terms of its symptoms, clinical assayresults, conditions, etc. As described herein, the Iowa strain of PRRSVhas been defined in terms of its histopathology and the clinicalsymptoms which it causes. Clinical analyses of the Iowa strain of PRRSVare described in detail in the Experiments below.

One then administers a prototype vaccine to a pig, then exposes the pigto the virus which causes the disease. This is known as “challenging”the pig and its immunological system. After observing the response ofthe challenged pig to exposure to the virus or infectious agent andanalyzing the ability of the prototype vaccine to protect the pig,efficacy studies are then performed by conventional, known methods. Apotency assay is then developed in a separate procedure by methods knownin the art, and prelicensing serials are then produced.

Prior to preparation of the prototype subunit vaccine (FIG. 1), theprotective or antigenic components of the vaccine virus should beidentified. Such protective or antigenic components include certainamino acid segments or fragments of the viral proteins (preferably coatproteins) which raise a particularly strong protective or immunologicalresponse in pigs; such antigenic protein fragments fused to non-PRRSVproteins which act as a carrier and/or adjuvant; single or multipleviral coat proteins themselves, oligomers thereof, and higher-orderassociations of the viral coat proteins which form virus substructuresor identifiable parts or units of such substructures; oligoglycosides,glycolipids or glycoproteins present on or near the surface of the virusor in viral substructures such as the nucleocapsid; lipoproteins orlipid groups associated with the virus, etc.

Antigenic amino acid segments or fragments are preferably at least 5amino acids in length, particularly preferably at least 10 amino acidsin length, and can be up to but not including the entire length of thenative protein. In the present invention, the binding affinity (orbinding constant or association constant) of an antigenic fragment ispreferably at least 1% and more preferably at least 10% of the bindingaffinity of the corresponding full-length protein (i.e., which isencoded by the same ORF) to a monoclonal antibody which specificallybinds the full-length protein. The monoclonal antibody whichspecifically binds to the full-length protein encoded by an ORF of aPRRSV is preferably deposited under the Budapest Treaty at an acceptabledepository, or is sequenced or otherwise characterized in terms of itsphysicochemical properties (e.g., antibody type [IgG, IgM, etc.],molecular weight, number of heavy and light chains, binding affinitiesto one or more known or sequenced proteins [e.g., selected from SEQ IDNOS:15, 17, 19, 21, 24, 26, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 67,69, 71, 73, 75 and 77], etc.).

Antigenic fragments of viral proteins (e.g., those encoded by one ormore of ORF's 2-6 of a PRRSV virus) are identified by methods known inthe art. For example, one can prepare polynucleic acids having atruncated ORF encoding a polypeptide with a predetermined number ofamino acid residues deleted from the N-terminus, C-terminus, or both.The truncated ORF can be expressed in vitro or in vivo in accordancewith known methods, and the corresponding truncated polypeptide can thenbe isolated in accordance with known methods. The immunoprotectiveproperties of the polypeptides may be measured directly (e.g., in vivo).Alternatively, the antigenic region(s) of the full-length polypeptidecan be determined indirectly by screening a series of truncatedpolypeptides against, for example, suitably deposited or characterizedmonoclonal antibodies. (If the alternative, indirect method isperformed, the failure of a truncated polypeptide to bind to aneutralizing monoclonal antibody is a strong indication that the portionof the full-length polypeptide deleted in the truncated polypeptidecontains an antigenic fragment.) Once identified, the antigenic orimmunoprotective portion(s) (the “subunit(s)”) of the viral proteins orof the virus itself may be subsequently cloned and/or purified inaccordance with known methods. (The viral/bacterial inactivation andsubunit purification protocols recited in FIG. 1 are optional.)Genetically engineered vaccines (FIG. 2) begin with a modification ofthe general procedure used for preparation of the other vaccines. Afterplaque-purification, the PRRS virus may be isolated from a suitabletissue homogenate by methods known in the art, preferably byconventional cell culture methods using PSP-36, ATCC CRL 11171 ormacrophage cells as hosts.

The RNA is extracted from the biologically pure virus by a known method,preferably by the guanidine isothiocyanate method using a commerciallyavailable RNA isolation kit (for example, the kit available fromStratagene, La Jolla, Calif.), and purified by one or more knownmethods, preferably by ultracentrifugation in a CsCl gradient. MessengerRNA may be further purified or enriched by oligo (dT)-cellulose columnchromatography.

The viral genome is then cloned into a suitable host by methods known inthe art (see Maniatis et al, “Molecular Cloning: A Laboratory Manual,”Cold Spring Harbor Laboratory (1989), Cold Spring Harbor, Mass.). Thevirus genome is then analyzed to determine essential regions of thegenome for producing antigenic portions of the virus. Thereafter, theprocedure for producing a genetically engineered vaccine is essentiallythe same as for a modified live vaccine, an inactivated vaccine or asubunit vaccine (see FIG. 1 of the present application and FIGS. 1-3 ofU.S. application Ser. No. 08/131,625). During prelicensing serials,expression of the cloned, recombinant subunit of a subunit vaccine maybe optimized by methods known to those in the art (see, for example,relevant sections of Maniatis et al, cited above).

The present vaccine protects pigs against a virus or infectious agentwhich causes a porcine reproductive and respiratory disease. Preferably,the present vaccine protects pigs against infection by PRRSV. However,the present vaccine is also expected to protect a pig against infectionby closely related variants of various strains of PRRSV as well.

Subunit virus vaccines may also be prepared from semi-purified virussubunits by the methods described above in the discussion of FIG. 1. Forexample, hemagglutinin isolated from influenza virus and neuraminidasesurface antigens isolated from influenza virus have been prepared, andshown to be less toxic than the whole virus. Subunit vaccines can alsobe prepared from highly purified subunits of the virus. An example inhumans is the 22-nm surface antigen of human hepatitis B virus. Humanherpes simplex virus subunits and many other examples of subunitvaccines for use in humans are known. Thus, methods of preparingpurified subunit vaccines from PRRSV cultured in a suitable host cellmay be applicable to the present subunit vaccine.

Attenuated virus vaccines can be found in nature and may havenaturally-occurring gene deletions (see Experiments VIII and IX below).Alternatively, attenuated vaccines may be prepared by a variety of knownmethods, such as serial passage (e.g., 5-25 times) in cell cultures ortissue cultures. However, the attenuated virus vaccines preferred in thepresent invention are those attenuated by recombinant gene deletions orgene mutations (as described above).

Genetically engineered vaccines are produced by techniques known tothose in the art. Such techniques include those using recombinant DNAand those using live viruses. For example, certain virus genes can beidentified which code for proteins responsible for inducing a strongerimmune or protective response in pigs. Such identified genes can becloned into protein expression vectors, such (but not limited to) as thebaculovirus vector (see, for example, O'Reilly et al, “BaculovirusExpression Vectors: A Lab Manual,” Freeman & Co. (1992)). The expressionvector containing the gene encoding the immunogenic virus protein can beused to infect appropriate host cells. The host cells are cultured, thusexpressing the desired vaccine proteins, which can be purified to adesired extent, then used to protect the pigs from a reproductive andrespiratory disease.

Genetically engineered proteins may be expressed, for example, in insectcells, yeast cells or mammalian cells. The genetically engineeredproteins, which may be purified and/or isolated by conventional methods,can be directly inoculated into animals to confer protection againstporcine reproductive and respiratory diseases. One or more envelopeproteins from a PRRSV (i.e., those encoded by ORF's 2-6) or antigenicportions thereof may be used in a vaccine to induce neutralizingantibodies. Nucleoproteins from a PRRSV may be used in a vaccine toinduce cellular immunity.

Preferably, the present invention transforms an insect cell line(HI-FIVE) with a transfer vector containing polynucleic acids obtainedfrom the Iowa strain of PRRSV. Preferably, the present transfer vectorcomprises linearized baculovirus DNA and a plasmid containing one ormore polynucleic acids obtained from the Iowa strain of PRRSV. The hostcell line may be co-transfected with the linearized baculovirus DNA anda plasmid, so that a recombinant baculovirus is made. Particularlypreferably, the present polynucleic acid encodes one or more proteins ofthe Iowa strain of PRRSV.

Alternatively, RNA or DNA from a PRRSV encoding one or more viralproteins (e.g., envelope and/or nucleoproteins) can be inserted intolive vectors, such as a poxvirus or an adenovirus, and used as avaccine.

Thus, the present invention further concerns a purified preparation of apolynucleic acid isolated from the genome of a PRRS virus, preferably apolynucleic acid isolated from the genome of the Iowa strain of PRRSV.The present polynucleic acid has utility (or usefulness) in theproduction of the present vaccine, in screening or identifying infectedor exposed animals, in identifying related viruses and/or infectiousagents, and as a vector for transforming cells and/or immunizing animals(e.g., pigs) with heterologous genes.

In the Experiments described hereinbelow, the isolation, cloning andsequencing of ORF's 2-7 of plaque-purified PRRSV isolate ISU-12(deposited on Oct. 30, 1992, in the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852, U.S.A., under the accessionnumbers VR 2385 [3×plaque-purified] and VR 2386 [non-plaque-purified])and ORF's 6-7 of PRRSV isolates ISU-22, ISU-55 and ISU-3927 (depositedon Sep. 29, 1993, in the American Type Culture Collection under theaccession numbers VR 2429, VR 2430 and VR 2431, respectively), ISU-79and ISU-1894 (deposited on Aug. 31, 1994, in the American Type CultureCollection under the accession numbers ______ and ______, respectively)are described in detail. However, the techniques used to isolate, cloneand sequence these genes can be also applied to the isolation, cloningand sequencing of the genomic polynucleic acids of any PRRSV. Thus, thepresent invention is not limited to the specific sequences disclosed inthe Experiments below.

For example, primers for making relatively large amounts of DNA by thepolymerase chain reaction (and if desired, for making RNA bytranscription-and/or protein by translation in accordance with known invivo or in vitro methods) can be designed on the basis of sequenceinformation where more than one sequence obtained from a PRRSV genomehas been determined (e.g., ORF's 2-5 of VR 2385 and Lelystad virus, orORF's 6-7 of VR 2385, VR 2429, VR 2430, ISU-79, ISU-1894, VR 2431 andLelystad virus). A region from about 15 to 50 nucleotides in lengthhaving at least 80% and preferably at least 90% identity is selectedfrom the determined sequences. A region where a deletion occurs in oneof the sequences (e.g., of at least 5 nucleotides) can be used as thebasis for preparing a selective primer for selective amplification ofthe polynucleic acid of one strain or type of PRRSV over another (e.g.,for the differential diagnosis of North American and European PRRSVstrains).

Once the genomic polynucleic acid is amplified and cloned into asuitable host by known methods, the clones can be screened with a probedesigned on the basis of the sequence information disclosed herein. Forexample, a region of from about 50 to about 500 nucleotides in length isselected on the basis of either a high degree of identity (e.g., atleast 90%) among two or more sequences (e.g., in ORF's 6-7 of the Iowastrains of PRRSV disclosed in Experiment III below), and apolynucleotide of suitable length and sequence identity can be preparedby known methods (such as automated synthesis, or restriction of asuitable fragment from a polynucleic acid containing the selectedregion, PCR amplification using primers which hybridize specifically tothe polynucleotide, and isolation by electrophoresis). Thepolynucleotide may be labeled with, for example, ³²P (for radiometricidentification) or biotin (for detection by fluorometry). The probe isthen hybridized with the polynucleic acids of the clones and detectedaccording to known methods.

The present Inventors have discovered that ORF 4 appears to be relatedto the virulence of PRRSV. For example, at least one isolate of PRRSVwhich shows relatively low virulence also appears to have a deletion inORF 4 (see, for example, Experiments VIII-XI below). Accordingly, in apreferred embodiment, the present invention is concerned with apolynucleic acid obtained from a PRRSV isolate which confers immunogenicprotection directly or indirectly against a subsequent challenge with aPRRSV, but in which ORF 4 is deleted or mutated to an extent which wouldrender a PRRSV containing the polynucleic acid either low-virulent(i.e., a “low virulence” (lv) phenotype; see the explanation below) ornon-virulent (a so-called “deletion mutant”). Preferably, ORF 4 isdeleted or mutated to an extent which would render a PRRS virusnon-virulent. However, it may be desirable to retain regions of a PRRSVORF 4 in the present polynucleic acid which (i) encode an antigenic,immunoprotective peptide fragment and (ii) would not confer virulence toa PRRS virus containing the polynucleic acid.

The present invention also encompasses a PRRSV per se in which ORF 4 isdeleted or mutated to an extent which renders it either low-virulent ornon-virulent (e.g., VR 2431). Such a virus is useful as a vaccine or asa vector for transforming a suitable host (e.g., MA-104, PSP 36, CRL11171, MARC-145 or porcine alveolar macrophage cells) with aheterologous gene. Preferred heterologous genes which may be expressedusing the present deletion mutant may include those encoding a proteinor an antigen other than a porcine reproductive and respiratory syndromevirus antigen (e.g., pseudorabies and/or swine influenza virus proteinsand/or polypeptide-containing antigens, a porcine growth hormone, etc.)or a polypeptide-based adjuvant (such as those discussed below for thepresent vaccine composition).

It may also be desirable in certain embodiments of the presentpolynucleic acid which contain, for example, the 3′-terminal region ofORF 3 (e.g., from 200 to 700 nucleotides in length), at least part ofwhich may overlap with the 5′-region of ORF 4. Similarly, where the3′-terminal region of ORF 4 may overlap with the 5′-terminal region ofORF 5, it may be desirable to retain the 5′-region of ORF 4which-overlaps with ORF 5.

The present Inventors have also discovered that ORF 5 in the PRRSVgenome appears to be related to replication of the virus in mammalianhost cells capable of sustaining a culture while infected with PRRSV.Accordingly, the present invention is also concerned with polynucleicacids obtained from a PRRSV genome in which ORF 5 may be present inmultiple copies (a so-called “overproduction mutant”). For example, thepresent polynucleic acid may contain at least two, and more preferably,from 2 to 10 copies of ORF 5 from a high-replication (hr) phenotypePRRSV isolate.

Interestingly, the PRRSV isolate ISU-12 has a surprisingly large numberof potential start codons (ATG/AUG sequences) near the 5′-terminus ofORF 5, possibly indicating alternate start sites of this gene (see SEQID NO:13). Thus, alternate forms of the protein encoded by ORF 5 of aPRRSV isolate may exist, particularly where alternate ORF's encode aprotein having a molecular weight similar to that determinedexperimentally (e.g., from about 150 to about 250 amino acids inlength). The most likely coding region for ORF 5 of ISU-12 (SEQ IDNO:14) is indicated in FIG. 7.

One can prepare deletion and overproduction mutants in accordance withknown methods. For example, one can prepare a-mutant polynucleic acidwhich contains a “silent” or degenerate change in the sequence of aregion encoding a polypeptide. By selecting and making an appropriatedegenerate mutation, one can substitute a polynucleic acid sequencerecognized by a known restriction enzyme. For example, if such a silent,degenerate mutation is made at one or two of the 3′-end of ORF 3 and the5′- and 3′-ends of ORF 4 and ORF 5, one can insert a syntheticpolynucleic acid (a so-called “cassette”) which may contain multiplecopies of ORF 5, multiple copies of a viral envelope protein or anantigenic fragment thereof. The “cassette” may be preceded by a suitableinitiation codon (ATG), and may be suitably terminated with atermination codon at the 3′-end (TAA, TAG or TGA).

Of course, an oligonucleotide sequence which does not encode apolypeptide may be inserted, or alternatively, no cassette may beinserted. By doing so, one may provide a so-called deletion mutant.

Thus, in one embodiment of the present invention, the polynucleic acidencodes one or more proteins, or antigenic regions thereof, of a PRRSV.Preferably, the present nucleic acid encodes at least one antigenicregion of a PRRSV membrane (envelope) protein. More preferably, thepresent polynucleic acid contains at least one copy of the ORF-5 genefrom a high virulence (hv) phenotype isolate of PRRSV (see thedescription of “hv phenotype” below) and a sufficiently long fragment,region or sequence of at least one of ORF-2, ORF-3, ORF-4, ORF-5 and/orORF-6 from the genome of a PRRSV isolate to encode an antigenic regionof the corresponding protein(s) and effectively stimulate immunologicalprotection against a subsequent challenge with an hv phenotype PRRSVisolate. Even more preferably, at least one entire envelope proteinencoded by ORF-2, ORF-3, ORF-5 and/or ORF-6 of a PRRSV is contained inthe present polynucleic acid, and the present polynucleic acid excludesa sufficiently long portion of ORF 4 from an hv PRRSV to render a PRRSVcontaining the same either low-virulent or non-virulent. Particularlypreferably, the present polynucleic acid excludes the entire region ofan hv PRRSV ORF 4 which does not overlap with the 3′-end of ORF 3 andthe 5′-end of ORF 5.

Most preferably, the polynucleic acid is isolated from the genome of anisolate of the Iowa strain of PRRSV (for example, VR 2385 (3×plaque-purified ISU-12), VR 2386 (non-plaque-purified ISU-12), VR 2428(ISU-51), VR 2429 (ISU-22), VR 2430 (ISU-55), VR 2431 (ISU-3927), ISU-79and/or ISU-1894.

A preferred embodiment of the present invention concerns a purifiedpreparation which may comprise, consist essentially of or consist of apolynucleic acid having a sequence of the formula (I):5′-α-β-γ-3′  (I)wherein α encodes at least one polypeptide or antigenic fragment thereofencoded by a polynucleotide selected from the group consisting of ORF 2and ORF 3 of an Iowa strain of PRRSV and regions thereof encoding theantigenic fragments; and β is either a covalent bond or a linkingpolynucleic acid which excludes a sufficiently long portion of ORF 4from an hv PRRSV to render the hv PRRSV either low-virulent ornon-virulent; and γ is at least one copy of an ORF 5 from an Iowa strainof PRRSV, preferably from a high replication (hr) phenotype.

Alternatively, the present invention may concern a purified preparationwhich may comprise, consist essentially of or consist of a polynucleicacid having a sequence of the formula (II):5′-γ-δ-ε-3′  (II)where γ is at least one copy of an ORF 5 from an Iowa strain of PRRSV,preferably from an hv PRRSV isolate; δ is either a covalent bond or alinking polynucleic acid which does not materially affect transcriptionand/or translation of the polynucleic acid; and ε encodes at least onepolypeptide or antigenic fragment thereof encoded by a polynucleotideselected from the group consisting of ORF 6 and ORF 7 of an Iowa strainof PRRSV and regions thereof encoding the antigenic fragments; and whenδ is a covalent bond, γ may have a 3′-end which excludes the regionoverlapping with the 5′-end of a corresponding ORF 6. Preferably, ε is apolynucleotide encoding at least an antigenic region of a proteinencoded by an ORF 6 of an Iowa strain of PRRSV, and more preferably,encodes at least a protein encoded by an ORF 6 of an Iowa strain ofPRRSV.

The present invention may also concern a purified preparation which maycomprise, consist essentially of or consist of a polynucleic acid havinga sequence of the formula (III):5′-γ-β-γ-δ-ε-3′  (III)where α, β, γ, δ and ε are as defined in formulas (I) and (II) above.Thus, the present polynucleic acid may be selected from the groupconsisting of, from 5′ to 3′:(ORF 5)_(n)   (IV)ζ-(ORF 5)_(n)   (V)(ORF 5)_(n)-η  (VI)ζ-(ORF 5)_(n)-η  (VII)where:

-   -   ζ is selected from the group consisting of ORF 2-, ORF 3-, ORF        4*-, ORF 2-ORF 3-, ORF 2-ORF 4*-, ORF 3-ORF 4*- and ORF 2-ORF        3-ORF 4*-; and    -   η is selected from the group consisting of -ORF 5*, -ORF 6, -ORF        7, -ORF 5*-ORF 6, -ORF 5*-ORF 7, -ORF 6-ORF 7 and -ORF 5*-ORF        6-ORF 7;    -   wherein ORF 2, ORF 3, ORF 6 and ORF 7 each encode a protein        encoded by the second, third, sixth and seventh open reading        frames of an Iowa strain of PRRSV, respectively; ORF 4* is a        region of a fourth open reading frame of an Iowa strain of PRRSV        which (i) encodes an antigenic, immunoprotective peptide        fragment and which (ii) does not confer virulence to a PRRSV        containing the polynucleic acid; ORF 5 is a fifth open reading        frame of an hv PRRSV isolate; ORF 5* is a region of a fifth open        reading frame of an Iowa strain of PRRSV which (i) encodes an        antigenic, immunoprotective peptide fragment and (ii) does not        confer virulence to a PRRSV containing the polynucleic acid, and        which may have a 3′-end which excludes the portion overlapping        with the 5′-end of a corresponding ORF 6; and n≧1.

The present polynucleic acid may also comprise, consist essentially ofor consist of combinations of the above sequences, either as a mixtureof polynucleotides or covalently linked in either a head-to-tail(sense-antisense) or head-to-head fashion. Polynucleic acidscomplementary to the above sequences and combinations thereof (antisensepolynucleic acid) are also encompassed by the present invention. Thus,in addition to possessing multiple or variant copies of ORF 5, thepresent polynucleic acid may also contain multiple or variant copies ofone or more of ORF's 1-3 and 6-7 and regions of ORF's 4-5 of Iowa strainPRRSV's.

The present invention may also concern polynucleic acids comprising,consisting essentially of or consisting of the open reading frame 1a and1b from a PRRSV isolate. Based on information regarding virusesevolutionally related to PRRSV, ORF 1a and 1b of PRRSV are believed toencode an RNA polymerase. ORF 1a and 1b are translated into a singleprotein by frameshifting. Preferably, the polynucleic acid from ORF 1aand 1b of a PRRSV isolate is obtained from an Iowa strain of PRRSV.

Similar to the methods described above and in the following Experimentsfor ORF's 2-7, one can prepare a library of recombinant clones (e.g.,using E. coli as a host) containing suitably prepared restrictionfragments of a PRRSV genome (e.g., inserted into an appropriate plasmidexpressible in the host). The clones are then screened with a suitableprobe (e.g, based on a conserved-sequence of ORF's 2-3; see, forexample, FIG. 22). Positive clones can then be selected and grown to anappropriate level. The polynucleic acids can then be isolated from thepositive clones in accordance with known methods. A suitable primer forPCR can then be designed and prepared as described above to amplify thedesired region of the polynucleic acid. The amplified polynucleic acidcan then be isolated and sequenced by known methods.

The present purified preparation may also contain a polynucleic acidselected from the group consisting of sequences having at least 97%sequence identity (or homology) with at least one ORF 7 of VR 2385, VR2430 and/or VR 2431; and sequences having at least 80% and preferably atleast 90% sequence identity (or homology) with at least one of ORF's 1-6of VR 2385, VR 2428, VR 2429, VR 2430 and/or VR 2431. Preferably, thepolynucleic acid excludes a sufficiently long region or portion of ORF 4of the hv PRRSV isolates VR 2385, VR 2429, ISU-28, ISU-79 and/or ISU-984to render the isolate low-virulent or non-virulent.

In the context of the present application, “homology” refers to thepercentage of identical nucleotide or amino acid residues in thesequences of two or more viruses, aligned in accordance with aconventional method for determining homology (e.g., the MACVECTOR orGENEWORKS computer programs, aligned in accordance with the proceduredescribed in Experiment III below).

Accordingly, a further aspect of the present invention encompasses anisolated polynucleic acid at least 90% homologous to a polynucleotidewhich encodes a protein, polypeptide or fragment thereof encoded byORF's 1-7 from an Iowa strain of PRRSV (e.g., SEQ ID NOS:15, 17, 19, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65 and 67). Preferably, thepresent isolated polynucleic acid encodes a protein, polypeptide, orantigenic fragment thereof which is at least 10 amino acids in lengthand in which amino acids non-essential for antigenicity may beconservatively substituted. An amino acid residue in a protein,polypeptide, or antigenic fragment thereof is conservatively substitutedif it is replaced with a member of its polarity group as defined below:

-   -   Basic Amino Acids:        -   lysine (Lys), arginine (Arg), histidine (His)    -   Acidic Amino Acids:        -   aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn),            glutamine (Gln)    -   Hydrophilic, Nonionic Amino Acids:        -   serine (Ser), threonine (Thr), cysteine (Cys), asparagine            (Asn), glutamine (Gln)    -   Sulfur-Containing Amino Acids:        -   cysteine (Cys), methionine (Met)    -   Hydrophobic, Aromatic Amino Acids:        -   phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp)    -   Hydrophobic, Nonaromatic Amino Acids:        -   glycine (Gly), alanine (Ala), valine (Val), leucine (Leu),            isoleucine (Ile), proline (Pro)

More particularly, the present polynucleic acid encodes one or more ofthe protein(s) encoded by the second, third, fourth, fifth, sixth and/orseventh open reading frames (ORF's 2-7) of the PRRSV isolates VR 2385,VR 2386, VR 2428, VR 2429, VR 2430, VR 2431, VR 2432, ISU-79 and/orISU-1894 (e.g., SEQ ID NOS:15, 17, 19, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63 and 65).

Relatively short-segments of polynucleic acid (about 20 bp or longer) inthe genome of a virus can be used to screen or identify tissue and/orbiological fluid samples from infected animals, and/or to identifyrelated viruses, by methods described herein and known to those ofordinary skill in the fields of veterinary and viral diagnostics andveterinary medicine. Accordingly, a further aspect of the presentinvention encompasses an isolated (and if desired, purified) polynucleicacid consisting essentially of a fragment of from 15 to 2000 bp,preferably from 18 to 1000 bp, and more preferably from 21 to 100 bp inlength, derived from ORF's 2-7 of a-PRRSV genome (preferably the Iowastrain of PRRSV). Particularly preferably, the present isolatedpolynucleic acid fragments are obtained from a terminus of one or moreof ORF's 2-7 of the genome of the Iowa strain of PRRSV, and mostpreferably, are selected from the group consisting of SEQ ID NOS:1-12,22 and 28-34.

The present invention also concerns a diagnostic kit for assaying aporcine reproductive and respiratory syndrome virus, comprising (a) afirst primer comprising a polynucleotide having a sequence of from 10 to50 nucleotides in length which hybridizes to a genomic polynucleic acidfrom an Iowa strain of porcine reproductive and respiratory syndromevirus at a temperature of from 25 to 75° C., (b) a second primercomprising a polynucleotide having a sequence of from 10 to 50nucleotides in length, said sequence of said second primer being foundin said genomic polynucleic acid from said Iowa strain of porcinereproductive and respiratory syndrome virus and being downstream fromthe sequence to which the first primer hybridizes, and (c) a reagentwhich enables detection of an amplified polynucleic acid. Preferably,the reagent is an intercalating dye, the fluorescent properties of whichchange upon intercalation into double-stranded DNA.

ORF's 6 and 7 are not likely candidates for controlling virulence andreplication phenotypes of PRRSV, as the nucleotide sequences of thesegenes are highly conserved among high virulence (hv) and low virulence(lv) isolates (see Experiment III below). However, ORF 5 in PRRSVisolates appears to be less conserved among high replication (hr) andlow replication (lr) isolates. Therefore, it is believed that thepresence of an ORF 5 from an hr PRRSV isolate in the present polynucleicacid will enhance the production and expression of a recombinant vaccineproduced from the polynucleic acid.

Accordingly, it is preferred that the present polynucleic acid, whenused for immunoprotective purposes (e.g., in the preparation of avaccine), contain at least one copy of ORF 5 from a high-replicationisolate (i.e., an isolate which grows to a titer of 10⁶-10⁷ TCID₅₀ in,for example, CRL 11171 cells; also see the discussions in ExperimentsVIII-XI below).

On the other hand, the lv isolate VR 2431 appears to be a deletionmutant, relative to hv isolates (see Experiments III and VIII-XI below).The deletion appears to be in ORF 4, based on Northern blot analysis.Accordingly, when used for immunoprotective purposes, the presentpolynucleic acid preferably does not contain a region of ORF 4 from anhv isolate responsible for its high virulence, and more preferably,excludes the region of ORF 4 which does not overlap with the adjacentORF's 3 and 5 (where ORF 4 overlaps with the adjacent ORF's 3 and 5).

It is also known (at least for PRRSV) that neither the nucleocapsidprotein nor antibodies thereto confer immunological protection againstthe virus (e.g., PRRSV) to pigs. Accordingly, the present polynucleicacid, when used for immunoprotective purposes, contains one or morecopies of one or more regions from ORF's 2, 3, 4, 5 and 6 of a PRRSVisolate encoding an antigenic region of the viral envelope protein, butwhich does not result in the symptoms or histopathological changesassociated with PRRS. Preferably, this region is immunologicallycross-reactive with antibodies to envelope proteins of other PRRSVisolates. Similarly, the protein encoded by the present immunoprotectivepolynucleic acid confers immunological protection to a pig administereda composition comprising the protein, and antibodies to this protein areimmunologically cross-reactive with the envelope proteins of other PRRSVisolates. More preferably, the present immunoprotective polynucleic acidencodes the entire envelope protein of a PRRSV isolate or a protein atleast 80% homologous thereto and in which non-homologous residues areconservatively substituted, or a protein at least 90% homologousthereto.

The present isolated polynucleic acid fragments can be obtained bydigestion of the cDNA corresponding to (complementary to) the viralpolynucleic acids with one or more appropriate restriction enzymes, canbe amplified by PCR and cloned, or can be synthesized using acommercially available automated polynucleotide synthesizer.

Another embodiment of the present invention concerns one or moreproteins or antigenic fragments thereof from a PRRS virus, preferablyfrom the Iowa strain of PRRSV. As described above, an antigenic fragmentof a protein from a PRRS virus (preferably from the Iowa strain ofPRRSV) is at least 5 amino acids in length, particularly preferably atleast 10 amino acids in length, and provides or stimulates animmunologically protective response in a pig administered a compositioncontaining the antigenic fragment.

Methods of determining the antigenic portion of a protein are known tothose of ordinary skill in the art (see the description above). Inaddition, one may also determine an essential antigenic fragment of aprotein by first showing that the full-length protein is antigenic in ahost animal (e.g., a pig). If the protein is still antigenic in thepresence of an antibody which specifically binds to a particular regionor sequence of the protein, then that region or sequence may benon-essential for immunoprotection. On the other hand, if the protein isno longer antigenic in the presence of an antibody which specificallybinds to a particular region or sequence of the protein, then thatregion or sequence is considered to be essential for antigenicity.

The present invention also concerns a′ protein or antigenic fragmentthereof encoded by one or more of the polynucleic acids defined above,and preferably by one or more of the ORF's of a PRRSV, more preferablyof the(Iowa strain of PRRSV. The present proteins and antigenicfragments are useful in immunizing pigs against PRRSV, in serologicaltests for screening pigs for exposure to or infection by PRRSV(particularly the Iowa strain of PRRSV), etc.

For example, the present protein may be selected from the groupconsisting of the proteins encoded by ORF's 2-7 of VR 2385, ISU-22 (VR2429), ISU-55 (VR 2430), ISU-1894, ISU-79 and ISU-3927 (VR 2431) (e.g.,SEQ ID NOS:15, 17, 19, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 67, 69and 71); antigenic regions of at least one of the proteins of SEQ ID SEQID NOS:15, 17, 19, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 67, 69 and 71having a length of from 5 amino acids to less than the full length ofthe polypeptides of SEQ ID NOS:15, 17, 19, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 67, 69 and 71; polypeptides at least 80% homologous with aprotein encoded by one of the ORF's 2-5 of VR 2385 (SEQ ID NOS:15, 67,69 and 71); and polypeptides at least 97% homologous with a proteinencoded by one of the ORF's.6-7 of VR 2385, VR 2429, VR 2430, ISU-1894,ISU-79 and VR 2431 (e.g., SEQ ID NOS:17, 19, 43, 45, 47, 49, 51, 53, 55,57, 59 and 61). Preferably, the present protein has a sequence selectedfrom the group consisting of SEQ ID NOS:15, 17, 19, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 67, 69 and 71; variants thereof which provideeffective immunological protection to a pig administered the same and inwhich from 1 to 100 (preferably from 1 to 50 and more preferably from 1to 25) deletions or conservative substitutions in the amino acidsequence exist; and antigenic fragments thereof at least 5 andpreferably at least 10 amino acids in length which provide effectiveimmunological protection to a pig administered the same.

More preferably, the present protein variant or protein fragment has abinding affinity (or association constant) of at least 1% and preferablyat least 10% of the binding affinity of the corresponding full-length,naturally-occurring protein to a monoclonal antibody which specificallybinds to the full-length, naturally-occurring protein (i.e., the proteinencoded by a PRRSV ORF). Most preferably, the present protein has asequence selected from the group consisting of SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NC:61, SEQ ID NO:67, SEQ ID NO:69 and SEQ ID NO:71.

The present invention may also concern a biologically pure virus,characterized in that it contains the present polynucleic acid and/orthat it causes a porcine reproductive and respiratory disease which mayinclude one or more of the following histological lesions: gross and/ormicroscopic lung lesions (e.g., lung consolidation), Type IIpneumocytes, myocarditis, encephalitis, alveolar exudate formation andsyncytia formation. The phrase “biologically pure” refers to a sample ofa virus or infectious agent in which all progeny are derived from asingle parent. Usually, a “biologically pure” virus sample is achievedby 3×plaque purification in cell culture.

In particular, the present biologically pure virus or infectious agentis an isolate of the Iowa strain of porcine reproductive and respiratorysyndrome virus, samples of which have been deposited under the terms ofthe Budapest Treaty at the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852, U.S.A., under the accessionnumbers VR 2385, VR 2386, VR 2428, VR 2429, VR 2430, VR 2431, ______ and______.

In addition to the characteristics (a)-(e) described above, the Iowastrain of PRRSV may also be characterized by Northern blots of its mRNA.For example, the Iowa strain of PRRSV may contain either 7 or 9 mRNA's,and may also have deletions or variations in their size. In particular,as will be described in the Experiments below, the mRNA's of the Iowastrain of PRRSV may contain up to four deletions, relative to VR 2385/VR2386.

The present invention further concerns a composition for protecting apig from viral infection, comprising an amount of the present vaccineeffective to raise an immunological response to a virus which causes aporcine reproductive and respiratory disease in a physiologicallyacceptable carrier.

An effective amount of the present vaccine is one in which a sufficientimmunological response to the vaccine is raised to protect a pig exposedto a virus which causes a porcine reproductive and respiratory diseaseor related illness. Preferably, the pig is protected to an extent inwhich from one to all of the adverse physiological symptoms or effects(e.g., lung lesions) of the disease to be prevented are found to besignificantly reduced.

The composition can be administered in a single dose, or in repeateddoses. Dosages may contain, for example, from 1 to 1,000 micrograms ofvirus-based antigen (vaccine), but should not contain an amount ofvirus-based antigen sufficient to result in an adverse reaction orphysiological symptoms of infection. Methods are known in the art fordetermining suitable dosages of active antigenic agent.

The composition containing the present vaccine may be administered inconjunction with an adjuvant or with an acceptable carrier which mayprolong or sustain the immunological response in the host animal. Anadjuvant is a substance that increases the immunological response to thepresent vaccine when combined therewith. The adjuvant may beadministered at the same time and at the same site as the vaccine or ata different time, for example, as a booster. Adjuvants also mayadvantageously be administered to the animal in a manner or at a site orlocation different from the manner, site or location in which thevaccine is administered. Adjuvants include aluminum hydroxide, aluminumpotassium sulfate, heat-labile or heat-stable enterotoxin isolated fromEscherichia coli, cholera toxin or the B subunit thereof, diphtheriatoxin, tetanus toxin, pertussis toxin, Freund's incomplete adjuvant,Freund's complete adjuvant, and the like. Toxin-based adjuvants, such asdiphtheria toxin, tetanus toxin and pertussis toxin, may be inactivatedprior to use, for example, by treatment with formaldehyde.

The present invention also concerns a method of protecting a pig frominfection against a virus which causes a porcine reproductive andrespiratory disease, comprising administering an effective amount of avaccine which raises an immunological response against such a virus to apig in need of protection against infection by such a virus. By“protecting a pig from infection” against a porcine reproductive andrespiratory syndrome virus or infectious agent, it is meant that afteradministration of the present vaccine to a pig, the pig shows reduced(less severe) or no clinical symptoms (such as fever) associated withthe corresponding disease, relative to control (infected) pigs. Theclinical symptoms may be quantified (e.g., fever, antibody count, and/orlung lesions), semi-quantified (e.g., severity of respiratory distress),or qualified.

The present invention concerns a system for measuring respiratorydistress in affected pigs. The present clinical respiratory scoringsystem evaluates the respiratory distress of affected pigs by thefollowing scale:

-   -   0=no disease; normal breathing    -   1=mild dyspnea and polypnea when the pigs are stressed (forced        to breathe in larger volumes and/or at an accelerated rate)    -   2=mild dyspnea and polypnea when the pigs are at rest    -   3=moderate dyspnea and polypnea when the pigs are stressed    -   4=moderate dyspnea and polypnea when the pigs are at rest    -   5=severe dyspnea and polypnea when the pigs are stressed    -   6=severe dyspnea and polypnea when the pigs are at rest

In the present clinical respiratory scoring system, a score of “0” isnormal, and indicates that the pig is unaffected by a porcinereproductive and respiratory disease. A score of “3” indicates moderaterespiratory disease, and a score of “6” indicates very severerespiratory disease. An amount of the present vaccine or composition maybe considered effective if a group of challenged pigs given the vaccineor composition show a lower average clinical respiratory score than agroup of identically challenged pigs not given the vaccine orcomposition. (A pig is considered “challenged” when exposed to aconcentration of an infectious agent sufficient to cause disease in anon-vaccinated animal.)

Preferably, the present vaccine composition is administered directly toa pig not yet exposed to a virus which causes a reproductive orrespiratory disease. The present vaccine may be administered orally orparenterally. Examples of parenteral routes of administration includeintradermal, intramuscular, intravenous, intraperitoneal, subcutaneousand intranasal routes of administration.

When administered as a solution, the present vaccine may be prepared inthe form of an aqueous solution, a syrup, an elixir, or a tincture. Suchformulations are known in the art, and are prepared by dissolution ofthe antigen and other appropriate additives in the appropriate solventsystems. Such solvents include water, saline, ethanol, ethylene glycol,glycerol, Al fluid, etc. Suitable additives known in the art includecertified dyes, flavors, sweeteners, and antimicrobial preservatives,such as thimerosal (sodium ethylmercurithiosalicylate). Such solutionsmay be stabilized, for example, by addition of partially hydrolyzedgelatin, sorbitol, or cell culture medium, and may be buffered bymethods known in the art, using reagents known in the art, such assodium hydrogen phosphate, sodium dihydrogen phosphate, potassiumhydrogen phosphate and/or potassium dihydrogen phosphate.

Liquid formulations may also include suspensions and emulsions. Thepreparation of suspensions, for example using a colloid mill, andemulsions, for example using a homogenizer, is known in the art.

Parenteral dosage forms, designed for injection into body fluid systems,require proper isotonicity and pH buffering to the corresponding levelsof porcine body fluids. Parenteral formulations must also be sterilizedprior to use.

Isotonicity can be adjusted with sodium chloride and other salts asneeded. Other solvents, such as ethanol or propylene glycol, can be usedto increase solubility of ingredients of the composition and stabilityof the solution. Further additives which can be used in the presentformulation include dextrose, conventional antioxidants and conventionalchelating agents, such as ethylenediamine tetraacetic acid (EDTA).

The present invention also concerns a method of producing the presentvaccine, comprising the steps of synthesizing or isolating a polynucleicacid of a PRRS virus (preferably the Iowa strain) encoding an antigenicprotein or portion thereof (preferably the viral coat protein),infecting a suitable host cell with the polynucleic acid, culturing thehost cell, and isolating the antigenic protein or portion thereof fromthe culture. Alternatively, the polynucleic acid itself can conferimmunoprotective activity to a host animal to which it is administered.

Preferably, the vaccine is collected from a culture medium by the stepsof (i) precipitating transfected, cultured host cells, (ii) lysing theprecipitated cells, and (iii) isolating the vaccine. Particularlypreferably, the host cells infected with the virus or infectious agentare cultured in a suitable medium prior to collecting.

Preferably, after culturing infected host cells, the infected host cellsare precipitated by adding a solution of a conventional poly(ethyleneglycol) (PEG) to the culture medium, in an amount sufficient toprecipitate the infected cells. The precipitated infected cells may befurther purified by centrifugation. The precipitated cells are thenlysed by methods known to those of ordinary skill in the art.Preferably, the cells are lysed by repeated freezing and thawing (threecycles of freezing and thawing is particularly preferred). Lysing theprecipitated cells releases the virus, which-may then be collected,preferably by centrifugation. The virus may be isolated and purified bycentrifuging in a CsCl gradient, then recovering the appropriatevirus-containing band from the CsCl gradient.

Alternatively, the infected cell culture may be frozen and thawed tolyse the cells. The frozen and thawed cell culture material may be useddirectly as a live vaccine. Preferably, however, the frozen and thawedcell culture material is lyophilized (for storage), then rehydrated foruse as a vaccine.

The culture media may contain buffered saline, essential nutrients andsuitable sources of carbon and nitrogen recognized in the art, inconcentrations sufficient to permit growth of virus-infected cells.Suitable culture media include Dulbecco's minimal essential medium(DMEM), Eagle's minimal essential medium (MEM), Ham's medium, medium199, fetal bovine serum, fetal calf serum, and other equivalent mediawhich support the growth of virus-infected cells. The culture medium maybe supplemented with fetal bovine serum (up to 10%) and/or L-glutamine(up to 2 mM), or other appropriate additives, such as conventionalgrowth supplements and/or antibiotics. A preferred medium is DMEM.

Preferably, the present vaccine is prepared from a virus or infectiousagent cultured in an appropriate cell line. The cell line is preferablyPSP-36 or an equivalent cell line capable of being infected with thevirus and cultured. An example of a cell line equivalent to PSP-36 isthe cell line PSP-36-SAH, which was deposited under the terms of theBudapest Treaty at the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852, U.S.A., on Oct. 28, 1992, under the depositnumber CRL 11171. Another equivalent cell line is MA-104, availablecommercially from Whittaker Bioproducts, Inc. (Walkersville, Md.).Preliminary results indicate that the Iowa strain of PRRSV can also becultured in porcine turbinate cells.

There also appears to be a relationship between the severity ofhistopathology caused by a challenge with a standard amount of aparticular isolate and the titer to which the isolate can be grown in amammalian host cell (e.g., CRL 11171, MA-104 cells [from African greenmonkey kidney], etc.).

Accordingly, the present invention also concerns a method of culturing aPRRS virus, comprising infecting cell line PSP-36, CRL 11171 or anequivalent cell line and culturing the infected cell line in a suitablemedium. An “equivalent cell line” to PSP-36 or CRL 11171 is one which iscapable of being infected with the virus and cultured, thereby producingculturable infected cells. Equivalent cell lines include MA-104,PSP-36-SAH and MARC-145 cells (available from the National VeterinaryServices Laboratory, Ames, Iowa), for example.

Preferably, the virus cultured is at least one isolate of the Iowastrain of PRRSV. Particularly preferably, the present vaccine isprepared from such a culture of the Iowa strain of PRRSV, cultivated inPSP-36 cells, and plaque-purified at least three times.

The cell line MA-104 is obtained from monkey kidney cells, and isepithelial-like. MA-104 cells form a confluent monolayer in cultureflasks containing Dulbecco's minimal essential medium and 10%. FBS(fetal bovine serum). When the monolayer is formed, the cells areinoculated with a sample of 10% homogenized tissue, taken from anappropriate tissue (such as lung and/or heart) in an infected pig.Preferably, appropriate antibiotics are present, to permit growth ofvirus and host cells and to suppress growth and/or viability of cellsother than the host cells (e.g., bacteria or yeast).

Both PSP-36 and MA-104 cells grow some isolates of the PRRS virus tohigh titers (over 10⁷ TCID₅₀/ml). PSP-36 and MA-104 cells will also growthe infectious agent associated with the Iowa strain of PRRSV. MA-104cells also are able to grow rotaviruses, polioviruses, and otherviruses.

CL2621 cells are believed to be of non-porcine origin and areepithelial-like, and are proprietary (Boehringer-Ingelheim). Bycontrast-to PSP-36 and MA-104, some samples of the virus which causesPRRS have been unsuccessfully cultured in CL2621 cells (Bautista et al,American Association of Swine Practitioners Newsletter, 4:32, 1992).

The primary characteristics of CL2621 are that it is of non-swineorigin, and is epithelial-like, growing in MEM medium. However, Benfieldet al (J. Vet. Diagn. Invest., 1992; 4:127-133) have reported thatCL2621 cells were used to propagate PRRS virus, but MA-104 cells wereused to control polio virus propagation, thus inferring that CL2621 isnot the same as MA-104, and that the same cell may not propagate bothviruses.

The Iowa strain of PRRSV generally cannot grow in cell lines other thanPSP-36, PSP-36-SAH and MA-104. As described above, however, some viruseswhich cause PRRS have been reported to grow in both CL2621 and primaryswine alveolar macrophages, although some strains of PRRS virus do notgrow in PSP-36, MA-104 or CL2621 cells.

The present vaccine, virus isolates, proteins and polynucleic acids canbe used to prepare antibodies which may provide immunological resistanceto a patient (in this case, a pig) exposed to a virus or infectiousagent. Antibodies encompassed by the present invention immunologicallybind either to (1) a vaccine which protects a pig against a PRRS virusor (2) to the PRRS virus itself. The present antibodies also have thefollowing utilities: (1) as a diagnostic agent for determining whether apig has been exposed to a PRRS virus or infectious agent, and (2) in thepreparation of the present vaccine. The present antibody may be used toprepare an immunoaffinity column by known methods, and theimmunoaffinity column can be used to isolate the virus or infectiousagent, or a protein thereof.

To raise antibodies to such vaccines or viruses, one immunizes anappropriate host animal, such as a mouse, rabbit, or other animals usedfor such inoculation, with the protein used to prepare the vaccine. Thehost animal is then immunized (injected) with one of the types ofvaccines described above, optionally administering an immune-enhancingagent (adjuvant), such as those described above. The host animal ispreferably subsequently immunized from 1 to 5 times at certain intervalsof time, preferably every 1 to 4 weeks, most preferably every 2 weeks.The host animals are then sacrificed, and their blood is collected. Serais then separated by known techniques from the whole blood collected.The sera contains antibodies to the vaccines. Antibodies can also bepurified by known methods to provide immunoglobulin G (IgG) antibodies.

The present invention also encompasses monoclonal antibodies to thepresent vaccines and/or viruses. Monoclonal antibodies may be producedby the method of Kohler et al (Nature, vol. 256 (1975), pages 495-497).Basically, the immune cells from a whole cell preparation of the spleenof the immunized host animal (described above) are fused with myelomacells by a conventional procedure to produce hybridomas. Hybridomas arecultured, and the resulting culture fluid is screened against the fluidor inoculum carrying the infectious agent (virus or vaccine).Introducing the hybridoma into the peritoneum of the host animalproduces a peritoneal growth of the hybridoma. Collection of the ascitesfluid of the host animal provides a sample of the monoclonal antibody tothe infectious agent produced by the hybridoma. Also, supernatant fromthe hybridoma cell culture can be used as a source of the monoclonalantibody, which is isolated by methods known to those of ordinary skillin the art. Preferably, the present antibody is of the IgG or IgM typeof immunoglobulin.

The present invention also concerns a method of treating a pig sufferingfrom a reproductive and respiratory disease, comprising administering aneffective amount of an antibody which immunologically binds to a viruswhich causes a porcine reproductive and respiratory disease or to avaccine which protects a pig against infection by a porcine reproductiveand respiratory virus in a physiologically acceptable carrier to a pigin need thereof.

The present method also concerns a method of diagnosing infection of apig by or exposure of a herd to a porcine reproductive and respiratorysyndrome virus and a diagnostic kit for assaying the same, comprisingthe present antibody (preferably a monoclonal antibody) and a diagnosticagent which indicates a positive immunological reaction with saidantibody (preferably comprising peroxidase-conjugated streptavidin, abiotinylated antibody to a PRRSV protein or antigen and a peroxidase).The present kit may further comprise aqueous hydrogen peroxide, aprotease which digests the porcine tissue sample, a fluorescent dye(e.g., 3,3′-diaminobenzidine tetrahydrochloride), and a tissue stain(e.g., hematoxylin).

A diagnosis of PRRS relies on compiling information from the clinicalhistory of the herd being diagnosed, from serology and pathology ofinfected pigs, and ultimately, on isolation of the PRRS virus (PRRSV)from the infected herd. Thus, the present method of detecting PRRSV isuseful in diagnosing infection by and/or exposure to the virus in aherd.

Clinical signs vary widely between farms, and thus, are not the mostreliable evidence of a definitive diagnosis, except in the case of asevere acute outbreak in naive herds which experience abortion storms,increased numbers of stillborn pigs, and severe neonatal and nursery pigpneumonia. Presently, the most common clinical presentation is pneumoniaand miscellaneous bacterial problems in 3-10 week old pigs. However,many PRRSV-positive herds have no apparent reproductive or respiratoryproblems.

There are some gross lesions that are very suggestive of PRRSV infectionin growing pigs. The most consistent experimentally reproducible grosslesion in 3-10 week-old pigs inoculated with several different PRRSVstrains is lymphadenopathy. In particular, iliac and mediastinal lymphnodes are often 3-10 times normal size, tan in color, and sometimescystic. The lymph nodes are not normally hyperemic, such as thelesion/conditions seen in bacterial septicemia.

Three histologic lesions are consistent with PRRSV infection.Interstitial pneumonia is commonly observed and is characterized byseptal infiltration with mononuclear cells, type 2 pneumocyteproliferation, and the presence of necrotic cells in the alveolarspaces. Nonsuppurative perivascular myocarditis and hyperplastic lymphnodes are commonly observed in the subacute stages of disease.

The degree of grossly visible pneumonia is strain dependent. In general,the lungs fail to collapse and have a patchy distribution of 10-80%tan-colored consolidation with irregular borders. Encephalitis is lessoften observed. Lesions in the fetus and placenta are rarely observed bylight microscopy.

However, the percentage of consolidation in the lungs provides aparticularly reliable test for infection by PRRSV (i.e., ≧10%consolidation at any time from 3 to 10 days post-infection (DPI) is apositive indication of infection), particularly by a high virulencephenotype (hv) virus (≧40% consolidation at any time from 3 to 10 daysDPI is a positive indication of infection by an hv PRRSV isolate).

In contrast to histopathology on lung tissue(s), most laboratories areroutinely using either an indirect-fluorescent antibody (IFA) test orimmunoperoxidase monolayer assay (IPMA) for serum antibody detection.With both the IFA and IPMA, one must subjectively determine endpointsand thus the tests are not automatable. Serum virus (SVN) neutralizationtests have also been developed, and ELISA tests are currently used insome research laboratories. Antibodies detected by the IFA test usuallyappear with 10 days of exposure but may be relatively short-lived,sometimes disappearing within 3 months.

Antibodies detected by ELISA usually appear within 3 weeks, but theirduration is unknown. SVN antibodies usually are not detected until 4-5weeks post exposure. The SVN test is considered less sensitive in acutedisease, but improvements have been made in the SVN test usingseronegative porcine serum supplementation. SVN titers reportedly aremeasurable longer than titers in IFA and IPMA, and thus, may be bettersuited for detection of positive animals in chronically infected herds.

In IFA, infected cells are fixed with acetone and methanol solutions,and antibodies for the convalescent sera of infected pigs are incubatedwith the infected cells, preferably for about 30 min. at 37° C. Apositive immunological reaction is one in which the antibody binds tothe virus-infected cells, but is not washed out by subsequent washingsteps (usually 3× with PBS buffer). A second antibody (an anti-antibody)labeled with a fluorescent reagent (FITC) is then added and incubated,preferably for anther 30 min. A positive immunological reaction resultsin the second antibody binding to the first, being retained afterwashing, and resulting in a fluorescent signal, which can be detectedand semi-quantified. A negative immunological reaction results in littleor no binding of the antibody to the infected cell. Therefore, thesecond, fluorescently-labeled antibody fails to bind, the fluorescentlabel is washed out, and little or no fluorescence is detected, comparedto an appropriate positive control.

IPA and ELISA kits are similar to the IFA kit, except that the secondantibody is labeled with a specific enzyme, instead of a fluorescentreagent. Thus, one adds an appropriate substrate for the enzyme bound tothe second antibody which results in the production of a coloredproduct, which is then detected and quantified by colorimetry, forexample.

Clinicians use antibody titers to determine the appropriate time forvaccination and/or implementation of management or control strategies.Prior to the present invention, serology tests did not provide antibodytiter levels adequate or reliable enough to make animal health caredecisions. It may have been appropriate to look for a change fromseronegative to seropositive status, or for at least a 4-fold increasein titer, as a positive indication of PRRSV infection/exposure. Lookingfor an increasing percentage of seropositive pigs in a particular agegroup over time in a herd can be useful to determine where the virus ismaintained and actively spreading. Sows infected in the early 3rdtrimester and aborting near term will likely not show increasing titers,however.

Virus isolation (VI) provides a definitive diagnosis, but has somelimitations. Virus is rarely isolated from stillborn or autolyzedaborted fetuses. Sows infected early in the last trimester may havetransient viremia and not abort until late term. Dead pigs of any ageare not the best samples for VI, because the virus does not survive wellat room temperature. Tissues should be removed from the carcass,packaged separately, and refrigerated as soon as possible to obtain aviable virus sample.

The best tissues for virus isolation are tonsil, lung, lymph nodes, andspleen. Serum is also an excellent sample for virus isolation, since (a)viremia is often prolonged in growing pigs, (b) the sample is easy tohandle, and (c) the sample can be quickly chilled and processed.

Variation between laboratories in the ability to isolate PRRSV is highbecause the tests, reagents, cell lines, and media used to detect/screenfor PRRSV have not been standardized. The efficacy of isolation variesbecause not all North American strains will grow on each cell line.Frozen tissue-section IFA tests have been used with limited success.

Serum virus neutralization (SVN) tests have also been developed, andELISA tests are currently used in some research laboratories. Antibodiesdetected by ELISA usually appear within 3 weeks, but their duration isunknown. SVN antibodies usually are not detected until 4-5 weekspost-exposure. The SVN test is considered less sensitive in acutedisease, but improvements have been made in the SVN test usingseronegative porcine serum supplementation. SVN titers reportedly aremeasurable for a longer period of time than titers in IFA and IPMA.Thus, SVN titers may be better suited for detection of positive animalsin chronically infected herds.

Prior to the present invention, however, serology tests did not provideantibody titer levels adequate or reliable enough to make animal healthcare decisions. Looking for an increasing percentage of seropositivepigs in a particular age group over time in a herd can also be useful todetermine where the virus is maintained and actively spreading. Sowsinfected in the early third trimester and aborting near term will likelynot show increasing titers, however. Thus, although it may have beenappropriate to look for a change from seronegative to seropositivestatus or for at least a 4-fold increase in titer as a positiveindication of PRRSV infection and/or exposure, a need for a morereliable titer-based assay is felt.

Thus, the present invention also concerns a method for detecting PRRSVantigen in tissues. The present diagnostic method, employing animmunoperoxidase test (IPT) preferably on formalin-fixed tissue, appearsto be quite useful to confirm the presence of active infection, and mayprovide a significant and meaningful increase in the reliability oftiter-based assays. A section of lungs, tonsils, mediastinal lymphnodes, and tracheobronchial lymph nodes from 26 pigs experimentallyinoculated with ATCC VR 2385 PRRSV was examined (see Experiment Vbelow). The virus was detected in 18/26 lungs, 26/26 tonsils, 15/26mediastinal lymph nodes, and 14/26 tracheobronchial lymph nodes. Thepigs in this study were killed over a 28 day period (post-inoculation).The virus was detected in at least one tissue in every pig necropsied upto 10 days post inoculation.

A complete technique for the present immunoperoxidase technique forPRRSV antigen detection in porcine tissues, based on astreptavidin-biotin assay, is described in Example V hereinunder.Briefly, the present method for detecting PRRSV comprises removingendogenous peroxidase from an isolated porcine tissue sample withaqueous hydrogen peroxide (preferably, a 0.1-5%, and more preferably,0.1-1.0% solution), then digesting the tissue with sufficient amount ofan appropriate protease to expose viral antigens (for example, ProteaseXIV, Sigma Chemical Company, St. Louis, Mo., and more preferably, a0.001-0.25% aqueous solution thereof). Thereafter, the method furthercomprises incubating primary monoclonal antibody ascites fluid(preferably diluted in TRIS/PBS by an amount of from 1:10 to 1:100,000,and more preferably, from 1:100 to 1:10,000) with the protease-treatedtissue sections in a humidified chamber for a sufficient length of timeand at an appropriate temperature to provide essentially completeimmunological binding to occur, if it can in fact occur (e.g., 16 hoursat 4° C.).

One suitable monoclonal antibody for use in the present diagnostic assayis SDOW-17 (available from Dr. David Benfield, South Dakota StateUniv.), which recognizes a conserved epitope of the PRRSV nucleocapsidprotein (Nelson et al, “Differentiation of U.S. and European isolates ofporcine reproductive and respiratory syndrome virus by monoclonalantibodies,” J. Clin. Micro., 31:3184-3189 (1993)).

The present method for detecting PRRSV then further comprises incubatingbiotinylated goat anti-mouse linking antibody (available from DakoCorporation, Carpintera, Calif.) with the tissue, followed by incubatingperoxidase-conjugated streptavidin with the biotinylatedantibody-treated tissue (Zymed Laboratories, South San Francisco,Calif.). The method then further comprises incubating theperoxidase-conjugated streptavidin-treated tissue with a chromagen, suchas 3,3′-diaminobenzidine tetrahydrochloride (available from VectorLaboratories Inc., Burlingame, Calif.), and finally, staining thetreated tissue with hematoxylin.

Particularly when combined with the further diagnostic techniques ofhistopathology, virus isolation procedures and serology, the presenttissue immunoperoxidase antigen detection technique offers a rapid andreliable diagnosis of PRRSV infection.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments, which are given forillustration of the invention, and are not intended to be limitingthereof.

EXPERIMENT I Molecular Cloning and Nucleotide Sequencing of the3′-Terminal Region of VR 2385 (Plaque-Purified ISU-12)

(I) Materials and Methods

(A) Virus Propagation and Purification

A continuous cell line, PSP-36, was used to isolate and propagateISU-12. The ISU-12 virus was plaque-purified 3 times on PSP-36 cells(plaque-purified ISU-12 virus was deposited under the terms andconditions of the Budapest Treaty at the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A., underAccession No. VR 2385). The PSP-36 cells were then infected with theplaque-purified virus. When more than 70% of the infected cells showedcytopathic changes, the culture was frozen and thawed three times. Theculture medium was then clarified by low-speed centrifugation at 5,000×gfor 15 min. at 4° C. The virus was then precipitated with 7% PEG-8000and 2.3% NaCl at 4° C. overnight with stirring, and the precipitate waspelleted by centrifugation. The virus pellets were then resuspended in 2ml of tris-EDTA buffer, and layered on top of a CsCl gradient(1.1245-1.2858 g/ml). After ultracentrifugation at 28,000 rpm for about8 hours at 20° C., a clear band with a density of 1.15-1.18 g/ml wasobserved and harvested. The infectivity titer of this band wasdetermined by IFA, and the titer was found to be 10⁶ TCID₅₀/ml. Typicalvirus particles were also observed by negative staining electronmicroscopy (EM).

(B) Isolation of Viral RNA

Total RNA was isolated from the virus-containing band in the CsClgradient with a commercially available RNA isolation kit (obtained fromStratagene). Poly(A) RNA was then enriched by oligo (dT)-cellulosecolumn chromatography according to the procedure described by themanufacturer of the column (Invitrogen).

(C) Construction of VR 2385 cDNA λ library

A general schematic procedure for the construction of a cDNA λ libraryis shown in FIG. 3. First strand cDNA synthesis from mRNA was conductedby reverse transcription using an oligo (dT) primer having a Xho Irestriction site. The nucleotide mixture contained normal DATP, dGTP,dTTP and the analog 5-methyl dCTP, which protects the cDNA fromrestriction enzymes used in subsequent cloning steps.

Second strand cDNA synthesis was then conducted with RNase H and DNApolymerase I. The cDNA termini were blunted (blunt-ended) with T4 DNApolymerase, ligated to EcoR I adaptors with T4 DNA ligase, andsubsequently phosphorylated with T4 polynucleotide kinase. The cDNA wasdigested with Xho I, and the digested cDNA were size-selected on anagarose gel. Digested cDNA larger than 1 kb in size were selected andpurified by a commercially available DNA purification kit (GENECLEAN,available from BIO 101, Inc., La Jolla, Calif.).

The purified cDNA was then ligated into lambda phage vector arms,engineered with Xho I and EcoR I cohesive ends. The ligated vector waspackaged into infectious lambda phages with lambda extracts. The SUREstrain (available from Stratagene) of E. coli cells were used fortransfection, and the lambda library was then amplified and titrated inthe XL-1 blue cell strain.

(D) Screening the X Library by Differential Hybridization

A general schematic procedure for identifying authentic clones of thePRRS virus VR 2385 strain by differential hybridization is shown in FIG.4, and is described hereunder. The λ library was plated on XL-1 bluecells, plaques were lifted onto nylon membranes in duplicates, anddenatured with 0.5 N NaOH by conventional methodology. Messenger RNA'sfrom both virus-infected PSP-36 cells and non-infected PSP-36 cells wereisolated by oligo (dT) cellulose column chromatography as described bythe manufacturer of the column (Invitrogen).

Complementary DNA probes were synthesized from mRNA's isolated fromvirus-infected PSP-36 cells and normal PSP-36 cells using random primersin the presence of ³²P-dCTP according to the procedure described by themanufacturer (Amersham). Two probes (the first synthesized fromvirus-infected PSP-36 cells, the other from normal, uninfected PSP-36cells) were then purified individually by Sephadex G-50 columnchromatography. The probes were hybridized with the duplicated nylonmembranes, respectively, at 42° C. in 50% formamide. Plaques whichhybridized with the probe prepared from virus infected cells, but notwith the probe prepared from normal cells, were isolated. The phagemidscontaining viral cDNA inserts were rescued by in vitro excision with thehelp of G408 helper phage. The rescued phagemids were then amplified onXL-1 blue cells. The plasmids containing viral cDNA inserts wereisolated by Qiagen column chromatography, and were subsequentlysequenced.

(E) Nucleotide Sequencing and Sequence Analysis

Plasmids containing viral cDNA inserts were purified by Qiagen columnchromatography, and sequenced by Sanger's dideoxy method with universaland reverse primers, as well as a variety of internal oligonucleotideprimers. Sequences were obtained from at least three separate clones.Additional clones or regions were sequenced when ambiguous sequence datawere obtained. The nucleotide sequence data were assembled and analyzedindependently using two computer software programs, GENEWORKS(IntelliGenetics, Inc., Mountain View, Calif.) and MACVECTOR(International Biotechnologies, Inc., New Haven, Conn.).

(F) Oligonucleotide Primers

Oligonucleotides were synthesized as single-stranded DNA using anautomated DNA synthesizer (Applied Biosystems) and purified by HPLC.Oligonucleotides PP284 (5′-CGGCCGTGTG GTTCTCGCCA AT-3′; SEQ ID NO:1) andPP285 (5′-CCCCATTTCC CTCTAGCGAC TG-3′; SEQ ID NO:2) were synthesized forPCR amplification. A DNA probe was generated with these two primers fromthe extreme 3′ end of the viral genome for Northern blot analysis (seediscussion below). Oligonucleotides PP286 (5′-GCCGCGGAAC CATCAAGCAC-3′;SEQ ID NO:3) and PP287 (5′-CAACTTGACG CTATGTGAGC-3′; SEQ ID NO:4) weresynthesized for PCR amplification. A DNA probe generated by these twoprimers was used to further screen the X library. Oligonucleotides PP288(5′-GCGGTCTGGA TTGACGACAG-3′; SEQ ID NO:5), PP289 (5′-GACTGCTAGGGCTTCTGCAC-3′; SEQ ID NO:6), PP386 (5′-GCCATTCAGC TCACATAGCG-3′; SEQ IDNO:7), PP286 and PP287 were used as sequencing primers to obtaininternal sequences.

(G) Northern Blot Analysis

A specific DNA fragment from the extreme 3′ end of the VR 2385 cDNAclone was amplified by PCR with primers PP284 and PP285. The DNAfragment was excised from an agarose gel with a commercially availableDNA purification kit (GENECLEAN, obtained from Bio 101), and labeledwith ³²P-dCTP by random primer extension (using a kit available fromAmersham). Total RNA was isolated from VR 2385-infected PSP-36 cells at36 hours post-infection, using a commercially available kit forisolation of total RNA according to the procedure described by themanufacturer (Stratagene). VR 2385 subgenomic mRNA species weredenatured with 6 M glyoxal and DMSO, and separated on a 1% agarose gel.(Results from a similar procedure substituting a 1.5% agarose gel aredescribed in Experiment II below and are shown in FIG. 5.) The separatedsubgenomic mRNA's were then transferred onto nylon membranes using aPOSIBLOT™ pressure blotter (Stratagene) Hybridization was carried out ina hybridization oven with roller bottles at 42° C. and 50% formamide.

Results

(A) Cloning, Identification and Sequencing of VR 2385 3′ Terminal Genome

An oligo (dT)-primed cDNA X library was constructed from a partiallypurified virus, obtained from VR 2385-infected PSP-36 cells. Problemswere encountered in screening the cDNA λ library with probes based onthe Lelystad virus sequence. Three sets of primers were prepared. Thefirst set (PP105 and PP106; SEQ ID NOS:8-9) correspond to positions14577 to 14596 and 14977 to 14995 of the Lelystad genomic sequence,located in the nucleocapsid gene region. The second set (PP106 andPP107, SEQ ID NOS:9-10) correspond to positions 14977 to 14995 and 14054to 14072 of the Lelystad genomic sequence, flanking ORF's 6 and 7. Thethird set (PM541 and PM542; SEQ ID NOS:11-12) correspond to positions11718 to 11737 and 11394 to 11413 of the Lelystad genomic sequence,located in the ORF-1b region. PP105: 5′ -CTCGTCAAGT ATGGCCGGT-3′ (SEQ IDNO:8) PP106: 5′ -GCCATTCGCC TGACTGTCA-3′ (SEQ ID NO:9) PP107:5′ -TTGACGAGGA CTTCGGCTG-3′ (SEQ ID NO:10) PM541: 5′ -GCTCTACCTGCAATTCTGTG-3′ (SEQ ID NO:11) PM542: 5′ -GTGTATAGGA CCGGCAACCG-3′ (SEQ IDNO:12)

All attempts to generate probes by PCR from the VR 2385 infectious agentusing these three sets of primers were unsuccessful. After severalattempts using the differential hybridization technique, however, theauthentic plaques representing VR 2385-specific cDNA were isolated usingprobes prepared from VR 2385-infected PSP-36 cells and normal PSP-36cells. The procedures involved in differential hybridization aredescribed and set forth in FIG. 4.

Three positive plaques (λ-4, λ-75 and λ-91) were initially identified.Phagemids containing viral cDNA inserts within the λ phage were rescuedby in vitro excision with the help of G408 helper phages. The inserts ofthe positive clones were analyzed by restriction enzyme digestion andterminal sequencing. The specificity of the cDNA clones was furtherconfirmed by hybridization with RNA from PSP-36 cells infected with theIowa strain of PRRSV, but not with RNA from normal PSP-36 cells. A DNAprobe was then generated from the 5′-end of clone λ-75 by PCR withprimers PP286 and PP287. Further positive plaques (λ-229, λ-268, λ-275,λ-281, λ-323 and λ-345) were identified using this probe. All λ cDNAclones used to obtain the 3′-terminal nucleotide sequences are presentedin FIG. 6. At least three separate clones were sequenced to eliminateany mistakes. In the case of any ambiguous sequence data, additionalclones and internal primers (PP288, PP289, PP286, PP287 and PP386) wereused to determine the sequence. The 2062-bp 3′-terminal sequence (SEQ IDNO:13) and the amino acid sequences encoded by ORF's 5, 6 and 7 (SEQ IDNOS:15, 17 and 19, respectively) are presented in FIG. 7.

(B) A Nested Set of Subgenomic mRNA

Total RNA from virus-infected PSP-36 cells was separated on 1%glyoxal/DMSO agarose gel, and blotted onto nylon membranes. A cDNA probewas generated by PCR with a set of primers (PP284 and PP285) flankingthe extreme 3′-terminal region of the viral genome. The probe contains a3′-nontranslational sequence and most of the ORF-7 sequence. Northernblot hybridization results show that the pattern of mRNA species fromPSP-36 cells infected with the Iowa strain of PRRSV is very similar tothat of Lelystad virus (LV), equine arteritis virus (EAV), lactatedehydrogenase-elevating virus (LDV) and coronavirus, in that virusreplication required the formation of subgenomic mRNA's.

The results also indicate that VR 2385-specific subgenomic mRNA'srepresent a 3′-nested set of mRNA's, because the Northern blot proberepresents only the extreme 3′ terminal sequence. The size of VR 2385viral genomic RNA (14 kb) and 6 subgenomic mRNA's (RNA 2 (3.0 kb), RNA 3(2.5 kb), RNA 4 (2.2 kb), RNA 5 (1.8 kb), RNA 6 (1.3 kb) and RNA 7 (0.98kb)) resemble those of LV, although there are differences in both thegenome and in subgenomic RNA species. Differences were also observed inthe relative amounts of the subgenomic mRNA's, RNA 7 being the mostpredominant subgenomic mRNA.

(C) Analysis of Open Reading Frames Encoded by Subgenomic RNA

Three large ORF's have been found in SEQ ID NO:13: ORF-5 (nucleotides[nt] 426-1025; SEQ ID NO:14), ORF 6 (nt 1013-1534; SEQ ID NO:16) and ORF7 (nt 1527-1895; SEQ ID NO:18). ORF 4, located at the 5′ end of theresulting sequence, is incomplete in the 2062-bp 3′-terminal sequence ofSEQ ID NO:13. ORF'S 5, 6 AND 7 each have a coding capacity of more than100 amino acids. ORF 5 and ORF 6 overlap each other by 13 bp, and ORF 6and ORF 7 overlap each other by 8 bp. Two smaller ORF's located entirelywithin ORF 7 have also been found, coding for only 37 aa and 43 aa,respectively. Another two short ORF's overlap fully with ORF 5. Thecoding capacity of these two ORF's is only 29 aa and 44 aa,respectively. No specific subgenomic mRNA's were correlated to thesesmaller ORF's by Northern blot analysis. ORF 6 and ORF 7 are believed toencode the viral membrane protein and capsid protein, respectively.

(D) Consensus Sequence for Leader Junction

Sequence analysis shows that a short sequence motif, AACC, may serve asthe site in the subgenomic mRNA's where the leader is added duringtranscription (the junction site). The junction site of ORF 6 is found21 bp upstream from the ATG start codon, and the junction site of ORF 7is found 13 bp upstream from the ATG start codon, respectively. No AACCconsensus sequence has been identified in ORF 5, although it has beenfound in ORF 5 of LV. Similar junction sequences have been found in LDVand EAV.

(E) 3′-Nontranslational Sequence and Poly (A) Tail

A 151 nucleotide-long (151 nt) nontranslational sequence following thestop codon of ORF 7 has been identified in the genome of VR 2385,compared to 114 nt in LV, 80 nt in LDV and 59 nt in EAV. The length ofthe poly (A) tail is at least 13 nucleotides. There is a consensussequence, CCGG/AAATT-poly (A) among PRRS virus VR 2385, LV and LDV inthe region adjacent to the-poly (A) tail.

(F) Sequence Comparison of VR 2385 and LV Genomes Among ORF's 5, 6 and7, and Among the Nontranslational Sequences

A comparison of the ORF-5 regions of the genomes of VR 2385 and of theLelystad virus (SEQ ID NO:20) is shown in FIG. 8. The correspondingcomparisons of the ORF-6 region, the ORF-7 region, and thenontranslational sequences of VR 2385 (SEQ ID NOS:16, 18 and 22,respectively) with the corresponding regions of LV (SEQ ID NOS:23, 25and 27, respectively) are shown in FIGS. 9, 10 and 11, respectively.

The results of the comparisons are presented in Table 1 below. Thenucleotide sequence homologies between LV and VR 2385 of the ORF 5, ORF6, ORF 7 and the nontranslational sequences are 53%, 78%, 58% and 58%,respectively.

The size of ORF 7 in LV is 15 nt larger than that in VR 2385. Also, the31-terminal nontranslational sequence is different in length (150 nt inVR 2385, but only 114 nt in LV). Like LV, the junction sequence, AACC,has also been identified in the genome of the Iowa strain of PRRS virusisolate VR 2385, except for ORF 5. The junction sequence of ORF 6 in VR2385 is 21 nt upstream from the ATG start codon, whereas the junctionsequence of ORF 6 is 28 nt upstream from ATG in LV. TABLE 1 Comparisonof genes of U.S. PRRSV isolate ATCC VR 2385 with those of Europeanisolate Lelystad virus* VR 2385 Lelystad Pred. Pred. Homology EstimatedSize N-glyco- protein Size N-glyco- protein between RNA size aminosylation size amino sylation size VR 2385 Gene RNA (in Kb) ORFs acidssites (kd) acids sites (kd) & Lelystad 5 5 1.9 5 200 2 22.2 201 2 22.453 6 6 1.4 6 174 1 19.1 173 2 18.9 78 7 7 0.9 7 123 2 13.6 128 1 13.8 58NTR — — — 151 — NA 114 0 NA 58 (nt) (nt) (nt)*Based on data presented by Conzelmann et al, Virology, 193, 329-339(1993), Meulenberg et al, Virology, 192, 62-72 (1993), and the resultspresented herein.

EXPERIMENT II The Expression of VR 2385 Genes in Insect Cells

(A) Production of Recombinant Baculovirus

The ORF-5, ORF-6 and ORF-7 sequences were individually amplified by PCRusing primers based on the VR 2385 (ISU-12) genomic nucleotide sequence.ORF-5 was amplified 10 using the following primers: (SEQ ID NO:28)5′ -GGGGATCCGG TATTTGGCAA TGTGTC-3′ (SEQ ID NO:29) 3′ -GGGAATTCGCCAAGAGCACC TTTTGTGG-5′

ORF-6 was amplified using the following primers: 5′ -GGGGATCCAGAGTTTCAGCG G-3′ (SEQ ID NO:30) 3′ -GGGAATTCTG GCACAGCTGA TTGAC-5′ (SEQID NO:31)

ORF-7 was amplified using the following primers: 5′ -GGGGATCCTTGTTAAATATG CC-3′ (SEQ ID NO:32) 3′ -GGGAATTCAC CACGCATTC-5′ (SEQ IDNO:33)

The amplified DNA fragments were cloned into baculovirus transfer vectorpVL1393 (available from Invitrogen). One μg of linearized baculovirusAcMNPV DNA (commercially available from Pharmingen, San Diego, Calif.)and 2 μg of PCR-amplified cloned cDNA-containing vector constructs weremixed with 50 μl of lipofectin (Gibco), and incubated at 22° C. for 15min. to prepare a transfection mixture.

One hour after seeding HI-FIVE cells, the medium was replaced with freshExcell 400 insect cell culture medium (available from JR ScientificCo.), and the transfection mixture was added drop by drop. The resultingmixture was incubated at 28° C. for six hours. Afterwards, thetransfection medium was removed, and fresh Excell 400 insect cellculture medium was added. The resulting mixture was then incubated at28° C.

Five days after transfection, the culture medium was collected andclarified. Ten-fold dilutions of supernatants were inoculated ontoHI-FIVE cells, and incubated for 60 min. at room temperature. After theinoculum was discarded, an overlay of 1.25% of agarose was applied ontothe cells. Incubation at 28° C. was conducted for four days. Thereafter,clear plaques were selected and picked using a sterile Pasteur pipette.Each plaque was mixed with 1 ml of Grace's insect medium into a 5 mlsnap cap tube, and-placed in-a refrigerator overnight to release thevirus from the agarose. Tubes were centrifuged for 30 minutes at 2000×gto remove agarose, and the supernatants were transferred into newsterile tubes. Plaque purification steps were repeated three times toavoid possible wild-type virus contamination. Pure recombinant cloneswere stored at −80° C. for further investigation.

(B) Expression of Recombinant Iowa Strain Infectious Agent Proteins

Indirect immunofluorescence assay and radioimmunoprecipitation testswere used to evaluate expression.

Indirect immunofluorescence assay: Hi-five insect cells in a 24-wellcell culture cluster plate were infected with wild-type baculovirus orrecombinant baculovirus, or were mock-infected. After 72 hours, cellswere fixed and stained with appropriate dilutions of swine anti-VR 2385polyclonal antibodies, followed by fluorescein isothiocyanate-labelled(FITC-labelled) anti-swine IgG. Immunofluorescence was detected in cellsinfected with the recombinant viruses, but not in mock-infected cells orcells inoculated with wild-type baculovirus. For example, FIG. 12 showsHI-FIVE cells infected with the recombinant baculovirus containing theVR 2385 ORF-7 gene (Baculo.PRRSV.7), which exhibit a cytopathic effect.Similar results were obtained with recombinant baculovirus containingORF-5 (Baculo.PRRSV.5) and ORF-6 (Baculo.PRRSV.6; data not shown). FIGS.13 and 14 show HI-FIVE cells infected with a recombinant baculoviruscontaining the VR 2385 ORF-6 gene and VR 2385 ORF-7 gene, respectively,stained with swine antisera to VR 2385, followed byfluorescein-conjugated anti-swine IgG, in which the insect cells areproducing recombinant Iowa strain viral protein. Similar results wereobtained with recombinant baculovirus containing ORF-5.

Radioimmunoprecipitation: Radioimmunoprecipitation was carried out witheach recombinant virus (Baculo.PRRSV.5, Baculo.PRRSV.6 andBaculo.PRRSV.7) to further determine the antigenicity and authenticityof the recombinant proteins. HI-FIVE insect cells were mock-infected, oralternatively, infected with each of the recombinant baculoviruses. Twodays after infection, methionine-free medium was added. Each mixture wasincubated for two hours, and then proteins labeled with ³⁵S-methionine(Amersham) were added, and the mixture was incubated for four additionalhours at 28° C. Radiolabeled cell lysates were prepared by three cyclesof freezing and thawing, and the cell lysates were incubated withpreimmune or immune anti-VR 2385 antisera. The immune complexes wereprecipitated with Protein A agarose and analyzed on SDS-PAGE afterboiling. X-ray film was exposed to the gels at −80° C., and developed.Bands of expected size were detected with ORF-6 (FIG. 15) and ORF-7(FIG. 16) products.

EXPERIMENT III

Summary:

The genetic variation and possible evolution of porcine reproductive andrespiratory syndrome virus (PRRSV) was determined by cloning andsequencing the putative membrane protein (M, ORF 6) and nucleocapsid (N,ORF 7) genes of six U.S. PRRSV isolates with differing virulence. Thededuced amino acid sequences of the putative M and N proteins from eachof these isolates were aligned with the corresponding sequences (to theextent known) of one other U.S. isolate, two European isolates, andother members of the proposed arterivirus group, including lactatedehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV).

The putative M and N genes displayed 96-100% amino acid sequenceidentity among U.S. PRRSV isolates with differing virulence. However,their amino acid sequences varied extensively from those of EuropeanPRRSV isolates, and displayed only 57-59% and 78-81% identity,respectively. The U.S. PRRSV isolates were more closely related to LDVthan were the European PRRSV isolates. The N protein of the U.S.isolates and European isolates shared about 50% and 40% amino acidsequence identity with that of LDV, respectively.

The phylogenetic dendrograms constructed on the basis of the putative Mand N genes of the proposed arterivirus group were similar and indicatedthat both U.S. and European PRRSV isolates were related to LDV and weredistantly related to EAV. The U.S. and European PRRSV isolates fell intotwo distinct groups with slightly different genetic distance relative toLDV. The results suggest that U.S. and European PRRSV isolates representtwo different genotypes, and that they may have evolved from LDV atdifferent time periods and have existed separately in U.S. and Europebefore their association with PRRS was recognized in swine.

ORF 6 encodes the membrane protein (M) of PRRSV, based on the similarcharacteristics of the ORF 6 of EAV, ORF 2 of LDV, and the M protein ofmouse hepatitis virus and infectious bronchitis virus (Meulenberg et al,Virology, 192, 62-72 (1993); Conzelmann et al, Virology, 193, 329-339(1993); Mardassi et al, Abstr. Conf. Res. Workers in Animal Diseases,Chicago, Ill., p. 43 (1993)). The product of ORF 7, the viralnucleocapsid protein (N), is extremely basic and hydrophilic (Meulenberget al, Virology, 192, 62-72 (1993); Conzelmann et al, Virology, 193,329-339 (1993); Murtaugh et al, Proc. Allen D. Leman Swine Conference,Minneapolis, Minn., pp. 43-45 (1993); Mardassi et al, Abstr. Conf. Res.Workers in Animal Diseases, Chicago, Ill., p. 43 (1993)).

The amino acid sequences encoded by ORF's 5, 6 and 7 of U.S. isolate VR2385 and of the European isolate Lelystad virus (LV) have been compared,and the identity (i.e., the percentage of amino acids in sequence whichare the same) between the two viruses is only 54%, 78% and 58%,respectively. Thus, striking genetic differences exist between the U.S.isolate VR 2385 and the European isolate LV (see U.S. application Ser.No. 08/131,625, filed Oct. 5, 1993).

However, the U.S. isolate VR 2385 is highly pathogenic compared toEuropean LV. Thus, PRRSV isolates in North America and in Europe appearto be antigenically and genetically heterogeneous, and differentgenotypes or serotypes of PRRSV may exist.

To further determine the genetic variation among the PRRSV isolates, theputative M and N genes of five additional U.S. PRRSV isolates withdiffering virulence were cloned and sequenced. Phylogenetic trees basedon the putative M and N genes of seven U.S. PRRSV isolates, two EuropeanPRRSV isolates and other members of the proposed arterivirus group,including LDV and EAV, have been constructed.

PRRSV isolates (ISU-12 (VR 2385/VR 2386), ISU-22 (VR 2429), ISU-55 (VR2430), ISU-79, ISU-1894 and ISU-3927 (VR 2431), each of which isdisclosed and described in U.S. application Ser. No. 08/131,625, filedOct. 5, 1993) were isolated from pig lungs obtained from different farmsin Iowa during PRRS outbreaks, according to the procedure described inU.S. application Ser. No. 08/131,625. A continuous cell line, ATCC CRL11171, was used to isolate and propagate these viruses. All viruses werebiologically cloned by three cycles of plaque purification prior topolynucleic acid sequencing.

Pathogenicity studies in caesarean-derived colostrum-deprived (CDCD)pigs, described in U.S. application Ser. No. 08/131,625, showed that VR2385, VR 2429 and ISU-79 were highly pathogenic, whereas VR 2430,ISU-1894 and VR 2431 were not as pathogenic. For example, VR 2385, VR2429 and ISU-79 produced from 50 to 800i consolidation of the lungtissues in experimentally-infected five-week-old CDCD pigs necropsied at10 days post inoculation, whereas VR 2430, ISU-1894 and VR 2431 producedonly 10 to 250 consolidation of lung tissues in the same experiment.

Experimental Section:

Monolayers of ATCC CRL 11171 cells were infected with each of the PRRSVisolates at the seventh passage at an m.o.i. of 0.1. Total cellular RNAwas isolated from infected cells by the guanidine isothiocyanate method(Sambrook et al, “Molecular Cloning: A Laboratory Manual,” 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)). The qualityof RNA from each isolate was determined by Northern blot hybridization(data not shown) with a cDNA probe generated from the extreme 3′-end ofthe VR 2385 genome by the polymerase chain reaction (PCR) with primersPP284 and PP285 (SEQ ID NOS: 1 AND 2), as described in U.S. applicationSer. No. 08/131,625. cDNA was synthesized from total cellular RNA withrandom primers using reverse transcriptase. The synthesized cDNA wasamplified by polymerase chain reaction (PCR) as described previously(Meng et al, J. Vet. Diagn. Invest., 5, 254-258 (1993)). Primers forRT-PCR were designed on the basis of a sequence in the genome of VR 2385which resulted in amplification of the entire protein coding-regions ofthe putative M and N genes (5′ primer: 5′-GGGGATCCAGAGTTTCAGCGG-3′ (SEQID NO:30); 3′ primer: 5′-GGGAATTCACCACGCATTC-3′ (SEQ ID NO:33)). Uniquerestriction sites (EcoR I and BamH I) at the termini of the PCR productswere introduced by conventional methods. A PCR product with the expectedsize of about 900 bp was obtained from each of the virus isolates.Southern blot hybridization was then used to confirm the specificity ofthe amplified products.

The ³²P-labelled cDNA probe from VR 2385 hybridized with the RT-PCRproducts from each of the above virus isolates. The PCR products of theputative M and N genes from each of the PRRSV isolates were purified andcloned into vector pSK+(Meng et al, J. Vet. Diagn. Invest. 5, 254-258(1993)). Plasmids containing the full length putative M and N genes weresequenced with an automated DNA Sequencer (obtained from AppliedBiosystems, Inc., Foster City Calif.). Three to four cDNA clones fromeach virus isolate were sequenced with universal and reverse primers, aswell as other virus specific sequencing primers (PP288:5′-GCGGTCTGGATTGACGAC-3′ (SEQ ID NO:5) and PP289:5′-GACTGCTAGGGCTTCTGC-3′ (SEQ ID NO:6), each of which is described inapplication Ser. No. 08/131,625, and DP966: 5′-AATGGGGCTTCTCCGG-3′ (SEQID NO:34)). The sequences were combined and analyzed by the MACVECTOR(International Biotechnologies, Inc.) and GENEWORKS (IntelliGenetics,Inc.) computer programs.

Analysis of the nucleotide sequences encoding the putative M and Nproteins of the 5 U.S. PRRSV isolates indicated that, like LV(Meulenberg et al, Virology, 192, 62-72 (1993)) and VR 2385, theputative M and N genes of each of the five additional U.S. isolatesoverlapped by 8 base pairs (bp). FIG. 17 shows the nucleotide sequenceof ORF's 6 and 7 of six U.S. PRRSV isolates and of LV, in which theISU-12 (VR 2385 and VR 2386) nucleotide sequence (SEQ ID NO:35) is shownfirst, and in subsequent sequences (SEQ ID NOS:36-41), only thosenucleotides which are different are indicated. Start codons areunderlined and indicated by (+1>), stop codons are indicated byasterisks (*), are indicated by (−), and the two larger deletions in theputative N gene are further indicated by ({circumflex over ( )})

FIGS. 18(A)-(B) show the alignment of amino acid sequences of theputative M (FIG. 18(A)) and N (FIG. 18(B)) genes of the proposedarterivirus group, performed with a GENEWORKS program (IntelliGenetics,Inc.), using the following parameters (default values): cost to open agap is 5, cost to lengthen a gap is 25, minimum diagonal length is 4,and maximum diagonal offset is 10. The EAV M gene sequence was omittedbecause the relatively low sequence identity with PRRSV and LDV requiresgaps in the alignments. The VR 2385/VR 2386 sequences (SEQ ID NOS:17 and19) are shown first, and in subsequent sequences (SEQ ID NOS:43, 45, 47,49, 51, 24, 53, 55, 57, 59, 61 and 26, respectively), only thedifferences are indicated. Deletions are indicated by (−), and the twolarger deletions in the putative N gene are further indicated by({circumflex over ( )}).

Numerous substitutions in the nucleotide sequence were distributedrandomly throughout the M and N genes in each of the five isolates, ascompared to VR 2385. Most of the substitutions are third base silentmutations when converted to amino acid sequences (see FIG. 18).Insertions and deletions are found in the nucleotide sequences of theputative M and N genes when comparing the U.S. isolates to LV, but notfound among the U.S. isolates (FIG. 17). For example, there are twolarger deletions, 15 and 10 nucleotides each, in the putative N gene ofthe U.S. isolates as compared to the LV N genome (FIG. 17).

The deduced amino acid sequences of the putative M and N genes from thesix Iowa strain PRRSV isolates are aligned with the corresponding Nsequence of another U.S. isolate, VR 2332 (Murtaugh et al, Proc. AllenD. Leman Swine Conference, Minneapolis, Minn., pp. 43-45 (1993)); twoEuropean PRRSV isolates, LV (Meulenberg et al, Virology 192, 62-72(1993)) and PRRSV isolate 10 (PRRSV-10) (Conzelmann et al, Virology,193, 329-339 (1993)); two LDV strains, LDV—C (Godney et al, Virology,177, 768-771 (1990)) and LDV—P (Kuo et al, Virus Res., 23, 55-72(1992)); and EAV (Den Boon et al, J. Virol., 65, 2910-2920 (1991)) (FIG.18).

The amino acid sequences of the putative N gene are highly conservedamong the seven U.S. PRRSV isolates (FIG. 18(B)), and displayed 96-100%amino acid sequence identity (Table 1). However, the putative N proteinsof the U.S. PRRSV isolates shared only 57-59% amino acid sequenceidentity with those of the two European isolates (Table 1), suggestingthat the U.S. and the European isolates may represent two differentgenotypes.

The putative M protein of each of the U.S. isolates was also highlyconserved, and displayed higher sequence similarity with the M proteinsof the two European isolates (FIG. 18(A)), ranging from 78 to 81% aminoacid identity (see Table 2 below). The putative N gene of each of theU.S. PRRSV isolates shared 49-50% amino acid sequence identity with thatof the LDV strains, whereas the two European PRRSV isolates shared only40-41l amino acid identity with that of the LDV strains (Table 2).

Two regions of amino acid sequence deletions, “KKSTAPM” (SEQ ID NO:62)and “ASQG” (SEQ ID NO:63), were found in the putative N proteins of eachof the seven U.S. PRRSV isolates, as well as the two LDV strains andEAV, when compared to the two European PRRSV isolates (FIG. 18(B)).These results indicated that the U.S. PRRSV isolates are more closelyrelated to LDV than are the European PRRSV isolates, and that PRRSV mayhave undergone divergent evolution in the U.S. and in Europe beforetheir association with PRRS was recognized in swine (Murtaugh, Proc.Allen D. Leman Swine Conference, Minneapolis, Minn., pp. 43-45 (1993)).

The European isolates may have diverged from LDV for a longer time thanthe U.S. isolates, and hence may have evolved first. However, the aminoacid sequence identity of the putative M gene between U.S. PRRSVisolates and LDV strains was similar to that between the European PRRSVisolates and LDV strains (Table 2). The putative M and N genes of theU.S. and European isolates of PRRSV shared only 15-17% and 22-24% aminoacid sequence identity with those of EAV, respectively.

The sequence homology of PRRSV with LDV and EAV suggests that theseviruses are closely related and may have evolved from a common ancestor(Plagemann et al, supra; Murtaugh, supra). The high sequenceconservation between LDV and PRRSV supported the hypothesis that PRRSVmay have evolved from LDV and was rapidly adapted to a new host species(Murtaugh, supra). Asymptomatic LDV infection were found in all strainsof mice (Murtaugh, supra; Kuo et al, supra). However, many pig forms areinfested with wild rodents (Hooper et al, J. Vet. Diagn. Invest., 6,13-15 (1994)), so it is possible that PRRSV evolved from LDV-infectedmice, and was rapidly adapted to a new host, swine.

The evolutionary relationships of PRRSV with other members of theproposed arterivirus group were determined on the basis of the aminoacid sequence of the-putative M and N genes. FIG. 19 shows aphylogenetic tree of the TABLE 2 Pairwise comparison of the amino acidsequences among the putative nucleocapsid and membrane proteins of theproposed arterivirus group VIRUS Virus VR2385 ISU-22 ISU-55 ISU-79ISU-1894 ISU-3927 VR2332 LV PRRSV-10 LDV-P LDV-C EAV VR2385 *** 98 96 9898 96 96 57 57 49 49 22 ISU-22 99 *** 98 100 100 98 98 57 57 49 49 23ISU-55 99 100 *** 98 98 97 96 59 59 49 49 23 ISU-79 98 99 99 *** 100 9898 57 57 49 49 23 ISU-1894 99 100 100 99 *** 98 98 57 57 49 49 23ISU-3927 96 97 97 97 97 *** 96 59 59 49 49 23 VR2332 N/A N/A N/A N/A N/AN/A *** 57 57 50 49 22 LV 78 79 79 79 79 81 N/A *** 99 41 40 23 PRRSV-1078 79 79 79 79 81 N/A 100 *** 41 40 23 LDV-P 50 51 51 51 51 51 N/A 53 53*** 98 23 LDV-C 49 50 50 50 50 50 N/A 52 52 96 *** 24 EAV 16 16 16 16 1615 N/A 17 17 16 17 ***Note.^(a)The values in the table are the percentage identity of amino acidsequences. N/A, not available.^(b)Nucleocapsid protein comparisons are presented in the upper righthalf and membrane protein comparisons are presented in the lower lefthalf.proposed arterivirus group based on the amino acid sequences of theputative M and N genes of this group. The phylogenetic tree for the Ngene is essentially the same as that for the M gene. The length of thehorizontal lines connecting one sequence to another is proportional tothe estimated genetic distance between sequences, as indicated by thenumbers given above each line. The UPGMA (unweighted pair group methodwith arithmetic mean) trees were constructed with a GENEWORKS program(IntelliGenetics, Inc.), which first clusters the two most similarsequences, then the average similarity of these two sequences isclustered with the next most similar sequences or subalignments, and theclustering continued in this manner until all sequences/isolates arelocated in the tree; both trees are unrooted.

The PRRSV isolates fall into two distinct groups. All the U.S. PRRSVisolates thus far sequenced are closely related and form one group. Thetwo European PRRSV isolates are closely related and form another group.Both the U.S. and European PRRSV isolates are related to LDV strains andare distantly related to EAV (FIG. 19).

The evolution patterns for the putative N and M genes also suggest thatPRRSV may be a variant of LDV. For example, the genetic distance of theU.S. PRRSV isolates is slightly closer to LDV than the European PRRSVisolates (FIG. 19), again suggesting that the U.S. and European PRRSVmay have evolved from LDV at different time periods and existedseparately before their association with PRRS was recognized in swine.European PRRSV may have evolved earlier than U.S. PRRSV. It is alsopossible that the U.S. and European PRRSV could have evolved separatelyfrom different LDV variants which existed separately in the U.S. andEurope.

A striking feature of RNA viruses is their rapid evolution, resulting inextensive sequence variation (Koonin et al, Critical Rev. Biochem. Mol.Biol., 28, 375-430 (1993)). Direct evidence for recombination betweendifferent positive-strand RNA viruses has been obtained (Lai, Microbiol.Rev., 56, 61-79 (1992)). Western equine encephalitis virus appears to bean evolutionally recent hybrid between Eastern equine encephalitis virusand another alphavirus closely related to Sindbis virus (Hahn et al,Proc. Natl. Acad. Sci. USA, 85, 5997-6001 (1988)). Accordingly, theemergence of PRRSV and its close relatedness to LDV and EAV is notsurprising. Although the capsid or nucleocapsid protein has been usedfor construction of evolutionary trees of many positive-strand RNAviruses, proteins with conserved sequence motifs such as RNA-dependentRNA polymerase, RNA replicase, etc., are typically more suitable forphylogenetic studies (Koonin et al, supra).

EXPERIMENT IV Cloning and Sequencing of cDNA Corresponding to ORF'S 2, 3and 4 of PRRSV VR 2385

The region including ORF's 2, 3, and 4 of the genome of the porcinereproductive and respiratory syndrome virus (PRRSV) isolate VR 2385 wascloned and analyzed. To clone the cDNA of PRRSV VR 2385, ATCC CRL 11171cells were infected with the virus at a m.o.i. of 0.1, and totalcellular RNA was isolated using an RNA Isolation Kit (Stratagene). ThemRNA fraction was purified through a Poly(A) Quick column (Stratagene),and the purified mRNA was used to generate a cDNA library. A cDNA oligodT library was constructed in Uni-ZAP XR λ vector using a ZAP-cDNAsynthesis kit (Stratagene), according to the supplier's instructions.Recombinant clones were isolated after screening of the library with anORF 4—specific hybridization probe (a 240 b.p. PCR product specific forthe 3′ end of ORF 4; SEQ ID NO:64). Recombinant pSK+containedPRRSV-specific cDNA was excised in vivo from positive λ plaquesaccording to the manufacturer's instructions.

Several recombinant plasmids with nested set of cDNA inserts with sizesranging from 2.3 to 3.9 kb were sequenced from the 5′ ends-of the clonedfragments. The nucleotide sequence of SEQ ID NO:65 was determined on atleast two independent cDNA clones and was 1800 nucleotides in length(FIG. 21). Computer analysis of the nucleotide and the deduced aminoacid sequences was performed using GENEWORKS (IntelliGenetics, Inc.) andMACVECTOR (International Biotechnologies, Inc.) programs.

Three partially overlapping ORF's (ORF 2, ORF 3 and ORF 4) wereidentified in this region. ORF's 2, 3 and 4 comprised nucleotides 12-779(SEQ ID NO:66), 635-1396 (SEQ ID NO:68) and 1180-1713 (SEQ ID NO:70),respectively, in the sequenced cDNA fragment.

A comparison of DNA sequences of ORF's 2, 3 and 4 of PRRSV VR 2385 withcorresponding ORF's of LV virus (SEQ ID NOS:72, 74 and 76, respectively)is presented in FIG. 22. The level of nucleotide sequence identity(homology) was 65′ for ORF 2, 64% for ORF 3 and 66i for ORF 4.

The predicted amino acid sequences encoded by ORF's 2-4 of PRRSV VR 2385(SEQ ID NOS:67, 69 and 71, respectively) and of LV (SEQ ID NOS:73, 75and 77, respectively) are shown in FIG. 23. A comparison of PRRSV VR2385 and LV shows a homology level of 58% for the protein encoded by ORF2, 55% for the protein encoded by ORF 3 and 66% for the protein encodedby ORF 4 (see FIG. 23).

EXPERIMENT V

An Immunoperoxidase Method of Detecting PRRSV

Four 3-week-old colostrum-deprived PRRSV negative animals wereinoculated intranasally with 10^(5.8) TCID₅₀ of PRRSV U.S. isolate ATCCVR 2386 propagated on ATCC CRL 11171 cells. These pigs were housed onelevated woven-wire decks and fed a commercial milk replacer. Two pigswere necropsied at 4 days post inoculation (DPI) and two at 8 DPI.

At the time of necropsy, the right and left lungs of each pig wereseparated and inflated via the primary bronchus with 45 ml of one offour fixatives and then immersion fixed for 24 hours. The fixatives usedin this experiment included 10% neutral buffered formalin, Bouin'ssolution, HISTOCHOICE (available from Ambresco, Solon, Ohio), and amixture containing 4% formaldehyde and 1% glutaraldehyde (4F:1G). Thetissues fixed in Bouin's were rinsed in five 30-minute changes of 70%ethyl alcohol after 4 hours fixation in Bouin's. All the tissues wereroutinely processed in an automated tissue processor beginning in 70%ethyl alcohol. Tissues were processed to paraffin blocks within 48 hoursof the necropsy.

Sections of 3 micron thickness were mounted on poly-1-lysine coatedglass slides, deparaffinized with two changes of xylene and rehydratedthrough graded alcohol baths to distilled water. Endogenous peroxidasewas removed by three 10-minute changes of 3% hydrogen peroxide. This wasfollowed by a wash-bottle rinse with 0.05 M TRIS buffer (pH 7.6)followed by a 5-minute TRIS bath. Protease digestion was performed onall tissue sections except those fixed in HISTOCHOICE. Digestion wasdone in 0.05% protease (Protease XIV, available from Sigma Chem., St.Louis, Mo.) in TRIS buffer for 2 minutes at 37° C. Digestion wasfollowed by a TRIS-buffer wash-bottle rinse and then a 5-minute coldTRIS buffer bath. Blocking for 20 minutes was done with a 5% solution ofnormal goat serum (available from Sigma Chem., St. Louis, Mo.).

The primary antibody used was the monoclonal antibody SDOW-17 (obtainedfrom Dr. David Benfield, South Dakota State Univ.), diluted 1:1000 inTRIS/PBS (1 part TRIS:9 parts PBS (0.01 M, pH 7.2)). The monoclonalantibody SDOW-17 recognizes a conserved epitope on the PRRSVnucleocapsid protein (Nelson et al, J. Clin. Microbiol., 31:3184-3189).The tissue sections were flooded with primary antibody and incubated at4° C. for 16 hours in a humidified chamber. The primary antibodyincubation was then followed by a wash-bottle rinse with TRIS buffer, a5-minute TRIS buffer bath, and then a 5-minute TRIS buffer bathcontaining 1% normal goat serum. The sections were flooded withbiotinylated goat anti-mouse antisera (obtained from Dako Corporation,Carpintera, Calif.) for 30 minutes. The linking antibody incubation wasfollowed by three rinses in TRIS buffer, as was done following primaryantibody incubation. The sections were then treated withperoxidase-conjugated streptavidin, diluted 1:200 in TRIS/PBS, for 40minutes, followed by a TRIS buffer wash-bottle rinse and a 5-minute TRISbuffer bath. The sections were then incubated with freshly-made3,3′-diaminobenzidine tetrahydrochloride (DAB, obtained from VectorLaboratories Inc., Burlingame, Calif.) for 8-10 minutes at roomtemperature, and then rinsed in a distilled water bath for 5 minutes.Counterstaining was done in hematoxylin (available from Shandon, Inc.,Pittsburgh, Pa.), and the sections were rinsed with Scott's Tap Water(10 g MgSO₄ and 2 g NaHCO₃ in 1 liter ultrapure water), then withdistilled water. After dehydration, the sections were covered withmounting media, and then a coverslip was applied.

Two negative controls were included. Substitution of TRIS/PBS buffer inplace of the primary antibody was done for one control. The othercontrol was done by substituting uninfected, age-matched, gnotobioticpig lungs for PRRSV-infected lungs.

Histological changes in infected tissues were characterized by moderatemultifocal proliferative interstitial pneumonia with pronounced type 2pneumocyte hypertrophy and hyperplasia, moderate infiltration ofalveolar septa with mononuclear cells, and abundant accumulation ofnecrotic cell debris and mixed inflammatory cells in the alveolarspaces. No bronchial or bronchiolar epithelial damage was observed.However, there was necrotic cell debris in the smaller airway lumina.

Intense and specific staining in the cytoplasm of infected cells wasobserved in the formalin- and Bouin's-fixed tissues. Staining was lessintense and specific in the 4F:1G-fixed tissues. There was poorstaining, poor cellular detail, and moderate background staining in theHISTOCHOICE-fixed tissues. Background staining was negligible with theother fixatives. Cellular detail was superior in the formalin-fixedtissue sections and adequate in the Bouin's- and 4F:1G-fixed tissues.

The labeled antigen was primarily within the cytoplasm of sloughed cellsand macrophages in the alveolar spaces (FIG. 24) and within cellulardebris in terminal airway lumina (FIG. 25). When compared to sectionsfrom the same block stained with hematoxylin and eosin, it wasdetermined that most of the labeled cells were macrophages, and somewere likely sloughed pneumocytes. Lesser intensities of staining wereobserved in mononuclear cells within the alveolar septa and rarely inhypertrophied type 2 pneumocytes.

Using an immunoperoxidase technique on frozen sections, others were ableto detect antigen in epithelial cells of brochioles and alveolar ductsas well as within cells in the alveolar septa and alveolar spaces (Polet al, “Pathological, ultrastructural, and immunohistochemical changescaused by Lelystad virus in experimentally induced infections of mysteryswine disease (synonym: porcine epidemic abortion and respiratorysyndrome (PEARS)),” Vet. Q., 13:137-143). We were unable to detectantigen in brochiolar epithelium using the present immunoperoxidasemethod.

The present streptavidin-biotin complex (ABC) technique using a PRRSVmonoclonal antibody can be modified as needed to identify PRRSV-infectedporcine lungs. Both 10% neutral-buffered formalin and Bouin's solutionare acceptable fixatives. Protease digestion enhances the antigendetection without destroying cellular detail. This technique istherefore quite useful for the diagnosis of PRRSV-induced pneumonia ofpigs, and for detection of PRRSV in lung tissue samples.

EXPERIMENT VI An Immunohistochemical Identification of Sites ofReplication of PRRSV

Summary: Four three-week-old caesarian-derived, colostrum-deprived(CDCD) pigs were inoculated intranasally with an isolate of porcinereproductive and respiratory syndrome virus. All inoculated pigsexhibited moderate respiratory disease. Two pigs were necropsied at 4days post inoculation (PI) and two at 9 days PI. Moderate consolidationof the lungs and severe enlargement of the lymph nodes were noted atnecropsy. Moderate perivascular lymphomacrophagic myocarditis wasobserved. Marked lymphoid follicular hyperplasia and necrosis wasobserved in the tonsil, spleen, and lymph nodes.

Porcine reproduction and respiratory syndrome virus antigen was detectedby the present streptavidin-biotin immunoperoxidase method primarilywithin alveolar macrophages in the lung and in endothelial cells andmacrophages in the heart. Macrophages and dendritic-like cells in thelymph nodes, spleen, tonsil, and thymus stained intensively positive forPRRSV nucleocapsid protein antigen as well.

Experimental section: Four pigs were snatched from the birth canal of asow that was positive for PRRSV antibody by indirect immunofluorescentantibody (IFA) examination of serum. The pigs were taken to a differentsite, housed on elevated woven-wire decks and raised on commercial milkreplacer. These pigs were bled at 0, 7, 14, and 21 days of age and foundto be negative for PRRSV antibody by the IFA test. No PRRSV was isolatedfrom the serum of the pigs or sow using MARC-145 cells (available fromNational Veterinary Services Laboratory, Ames, Iowa).

All four pigs were inoculated intranasally at 3 weeks of age with10^(5.8) TCID₅₀ of PRRSV U.S. isolate ATCC VR 2385 propagated on ATCCCRL 11171 cells. Mild-to-moderate respiratory disease was observed from3-9 days post inoculation (DPI). Two pigs were necropsied at 4 DPI andtwo at 9 DPI. At 4 DPI, one pig evidenced 31% and the other 36%tan-colored consolidation of the lungs. At 9 DPI, the remaining two pigsevidenced 37% and 46% consolidation of the lungs, respectively. Lymphnodes were moderately enlarged and edematous.

Lymphoid tissues collected at necropsy included the tonsil, thymus,spleen, tracheobronchial, mediastinal, and medial iliac lymph nodes.Lymphoid tissues were fixed by immersion for 24 hours in 10% neutralbuffered formalin, processed routinely in an automated tissue processor,embedded in paraffin, sectioned at 6 microns and stained withhematoxylin and eosin. Additional sections (including the lung tissuesections above) were cut at 3 microns and mounted on poly-L-lysinecoated slides for immunohistochemistry.

The immunoperoxidase assay described in Experiment VI above wasrepeated. Briefly, after endogenous peroxidase was removed with 3%hydrogen peroxide, primary monoclonal antibody ascites fluid diluted1:1000 in TRIS/PBS was added for 16 hours at 4° C. in a humidifiedchamber. The monoclonal antibody SDOW-17 (obtained from Dr. DavidBenfield, South Dakota State Univ.), which recognizes a conservedepitope of the PRRSV nucleocapsid protein, was used. Biotinylated goatanti-mouse linking antibody (obtained from Dako Corporation, Carpintera,Calif.) was added, followed by treatment with peroxidase-conjugatedstreptavidin (obtained from Zymed Laboratories, South San Francisco,Calif.) and incubation with 3,3′-diaminobenzidine tetrahydrochloride(obtained from Vector Laboratories Inc., Burlingame, Calif.). Theincubated sample was finally counterstained in hematoxylin.

Microscopic lesions included interstitial pneumonia, myocarditis,tonsillitis, and lymphadenopathy. One section of lung from each lobe wasexamined. The interstitial pneumonic lesions were characterized byseptal infiltration with mononuclear cells, hyperplasia and hypertrophyof type 2 pneumocytes, and accumulation of macrophages and necrotic celldebris in alveolar spaces. These lesions were moderate and multifocal by4 DPI and severe and diffuse by 9 DPI. Bronchi and bronchiolarepithelium was unaffected. PRRSV antigen was readily detected byimmunohistochemistry in alveolar macrophages. Large dark-brown PRRSVantigen-positive macrophages were often found in groups of 5-10 cells. Afew PRRSV antigen-positive mononuclear cells were observed within thealveolar septa. PRRSV antigen was not detected in any tissues of thenegative control pigs.

One section of left and one section of right ventricle were examined. At4 DPI, there were small, randomly distributed, perivascular foci oflymphocytes and macrophages. There was moderate multifocal perivascularlymphoplasmacytic and histiocytic inflammation by 9 DPI. Moderatenumbers or endothelial cells lining small capillaries of lymphaticsthroughout the myocardium stained strongly positive for PRRSV antigen(FIG. 26) at both 4 and 9 DPI. The PRRSV antigen-positive endothelialcells frequently were not surrounded by inflammatory cells at 4 DPI, butwere in areas of inflammation at 9 DPI. A few macrophages betweenmyocytes and in perivascular areolar tissue also stained stronglypositive for PRRSV antigen.

A mild tonsillitis with necrosis was observed. Necrotic foci of 1-10cells with pyknosis and karyorrhexis were commonly observed in thecenter of prominent follicles and less often in the surroundinglymphoreticular tissue. Large numbers of lymphocytes and macrophageswere observed within the crypt epithelium, and moderate amounts ofnecrotic cell debris were observed in crypts. PRRSV antigen was readilydetected within cells in the center of hyperplastic follicles, in thesurrounding lymphoreticular tissue, and within cells in the cryptepithelium (FIG. 27). Staining was also present amongst necrotic debrisin the crypts. In all these sites, the PRRSV antigen-positive cellsresembled macrophages or dendritic-like cells.

Thymic lesions were minimal. There were a few necrotic foci withpyknosis and karyorrhexis in the medulla. These foci tended to involveor be near thymic corpuscles. PRRSV antigen was frequently identifiedwithin macrophages near these necrotic areas and less often within largeisolated macrophages in the cortex.

Necrotic foci and single necrotic cells were evident with germinalcenters of lymphoid nodules and in periarteriolar lymphoid sheaths(PALS) of the spleen. PRRSV antigen positive staining cells wereconcentrated in the center of lymphoid follicles and scatteredthroughout PALS. The positive cells generally had large oval nuclei andabundant cytoplasm with prominent cytoplasmic projections, compatiblewith macrophages or dendritic cells. Lesser numbers of positive-stainingfusiform-shaped cells in the marginal zone were observed. The size andlocation of these cells suggests that they are reticular cells.

The predominant lymph node changes were subcapsular edema, foci ofnecrosis in lymphoid follicles, and the presence of syncytial cells atthe border of the central lymphoid tissue with the loose peripheralconnective tissue. The high endothelial venules were unusually prominentand often swollen. The syncytial cells had 2-10 nuclei with multipleprominent nucleoli and moderate eosinophilic cytoplasm. These cells didnot appear to contain PRRSV antigen. Intense and specific cellularcytoplasmic staining was observed in the follicles. The positive cellshad large nuclei with abundant cytoplasm and prominent cytoplasmicprocesses (FIG. 27). These cells resembled macrophages or dendriticcells. Lesser numbers of positive cells were observed in theperifollicular lymphoid tissue.

The lesion severity and the amount of antigen detected within varioustissues was generally similar at 4 and 9 DPI. The gross size of thelymph nodes and the number of syncytial cells in lymph nodes were moreprominent at 9 DPI than at 4 DPI. The amount of antigen detected in theheart was also greater at 9 DPI.

Tissues from age-matched uninfected CDCD pigs were used for histologicand immunohistochemical controls. Other negative controls forimmunohistochemistry included using the same protocol less the primaryPRRSV antibody on the infected pig tissues. PRRSV antigen was notdetected in any of the negative controls.

Conclusions: The immunohistochemical procedure described herein isuseful for detecting PRRSV antigen in the lung, heart and lymphoidtissues of PRRSV-infected pigs. Severe interstitial pneumonia andmoderate multifocal perivascular lymphohistiocytic myocarditis wasobserved. Marked lymphoid follicular hyperplasia and necrosis ofindividual or small clusters of cells in the tonsil, spleen, and lymphnodes was also observed. PRRSV antigen was readily detected in alveolarmacrophages in the lung and in endothelial cells and macrophages in theheart.

Macrophages and dendritic-like cells in tonsil, lymph nodes, thymus, andspleen stained intensely positive for viral antigen as well.

PRRSV may replicate in the tonsil with subsequent viremia and furtherreplication, primarily within macrophages in the respiratory andlymphoid systems of the pig.

EXPERIMENT VII

Diagnosing PRRS:

The present streptavidin-biotin immunoperoxidase test for detection ofPRRSV antigen in tissues is quite useful to confirm the presence ofactive infection. 26 pigs were experimentally inoculated with ATCC VR2385 PRRSV in accordance with the procedure in Experiments V/VI above.One section of each of the lungs, tonsils, mediastinal lymph nodes, andtracheobronchial lymph nodes from each pig was examined. The virus wasdetected by the immunoperoxidase assay of Experiment V in 23/26 lungs,26/26 tonsils, 15/26 mediastinal lymph nodes, and 14/26 tracheobronchiallymph nodes.

The pigs in this experiment were killed over a 28 day periodpost-inoculation. The virus was detected in at least one tissue in everypig necropsied up to 10 days post inoculation.

A complete technique for the streptavidin-biotin based immunoperoxidasetechnique for PRRSV antigen detection in porcine tissues is described inExperiment V infra. Briefly, after endogenous peroxidase removal by 3%hydrogen peroxide and digestion with 0.05% protease (Protease XIV, SigmaChemical Company, St. Louis, Mo.), primary monoclonal antibody ascitesfluid diluted 1:1000 in TRIS/PBS is added for 16 hours at 4° C. in ahumidified chamber. The monoclonal antibody used was SDOW-17 (Dr. DavidBenfield, South Dakota State Univ.), which recognizes a conservedepitope of the PRRSV nucleocapsid protein (Nelson et al,“Differentiation of U.S. and European isolates of porcine reproductiveand respiratory syndrome virus by monoclonal antibodies,” J. Clin.Micro., 31:3184-3189 (1993)). Biotinylated goat anti-mouse linkingantibody (Dako Corporation, Carpintera, Calif.) is then contacted withthe tissue, followed by treatment with peroxidase-conjugatedstreptavidin (Zymed Laboratories, South San Francisco, Calif.),incubation with 3,3′-diaminobenzidine tetrahydrochloride (VectorLaboratories Inc., Burlingame, Calif.), and finally staining withhematoxylin.

Particularly when combined with one or more additional analyticaltechniques such as histopathology, virus isolation and/or serology, thepresent tissue immunoperoxidase antigen detection assay offers a rapidand reliable diagnosis of PRRSV infection.

EXPERIMENT VIII

The pathogenicity of PRRSV isolates in 4-8 week old pigs was determined.The isolates were divided into two groups: (1) phenotypes with highvirulence (hv) and (2) phenotypes with low virulence (lv) (see Table 3below). For example, the mean percentage of lung consolidation of groupsof pigs inoculated with a PRRSV isolate is shown in Table 4 below. Thepathogenicity of a number of PRRSV isolates at 10 DPI is shown in Table5 below. The results in Table 5 were statistically analyzed to verifythe difference between hv and lv phenotypes, as determined by percentagelung consolidation.

Isolates characterized as high virulence produce severe clinical diseasewith high fever and dyspnea. In general, hv isolates produce severepneumonia characterized by proliferative interstitial pneumonia withmarked type II pneumocyte proliferation, syncytial cell formation,alveolar exudate accumulation, mild septal infiltration with mononuclearcells, encephalitis and myocarditis (designated PRRS-B hereinafter).Isolates characterized as low virulence do not produce significantclinical disease and produce mild pneumonia characterized predominatelyby interstitial pneumonia with septal infiltration by mononuclear cells,typical of classical PRRS (designated PRRS-A hereinafter). TABLE 3Characteristics and Pathogenicity of PRRSV Isolates MicroscopicLesions** Severity of Lesion No. of gross Type Virus Subgenomicpneumonia* in Isolate mRNAs mRNA 4 lesions Lung Heart Brain HighVirulence (hv) VR 2385 6 Normal ++++ B ++++ ++++ VR 2429 8 Normal ++++ B++++ +++ ISU-28 ND ND +++ B ++++ ++++ ISU-79 8 Normal ++++ B +++ +++ISU-984 ND ND +++ B +++ +++ Low Virulence (lv) ISU-51 ND ND + A + + VR2430 8 Normal + A/B + + ISU-95 ND ND + A + + ISU-1894 6 Normal + A/B + +VR 2431 6 Deletion + A/B − − Lelystad*** 6 Normal + A +/− +/−*(−) normal, (+) mild, (++) moderate, (+++) severe, (++++) very severepneumonia.**PRRSV isolates produce two types of microscopic lung lesions: Type Alesions include interstitial pneumonia with mild septal infiltrationwith mononuclear cells typical of PRRS as described by Collins et# al (1992); Type B lesions include proliferation of type IIpneumocytes, and are typical of those described as PIP (Halbur et al1993).***Pol et al, (Vet. Quart., 13: 137-143 (1991); Wensvoort et al,Antigenic comparison of Lelystad virus and swine infertility andrespiratory syndrome virus. J. Vet. Diagn. Invest., 4: 134-138 (1992);Meulenberg et al,# Lelystad virus, the causative agent of porcine epidemic abortion andrespiratory syndrome (PEARS), is related to LDV and EAV. Virology, 192:62-72 (1993).

TABLE 4 Mean % Lung Consolidation Score at VIRUS DPI* ISOLATE 3 10 21 28VR-2385 29 77.3 37.3 6.0 VR-2386pp 20.5 77.5 25.0 0.0 ISU-22 26.5 64.836.5 11.0 ISU-984 7.25 76.0 21.0 0.5 ISU-3927 13.5 10.5 0 0.0 PSP-36 0 00 0.0 UNINOC 0 0 0 0.0*Score range is from 0-100% consolidation of the lung tissue.

TABLE 5 Mean % Lung Consolidation INOCULUM NO. PIGS at 10 DPI ± S.D.Uninfected 10 0 ± 0 CRL 11171 Cell Line 10 0 ± 0 ISU-51 10 16.7 ± 9.0 ISU-55 10 20.8 ± 15.1 ISU-1894 10 27.4 ± 11.7 ISU-79 10 51.9 ± 13.5VR-2386pp 10 54.3 ± 9.8  ISU-28 10 62.4 ± 20.9*Pathogenicity of PRRSV isolates ISU-28, VR 2386pp and ISU-79 were notsignificantly different (p > 0.05) from each other, but were differentfrom that of ISU-51, ISU-55, and ISU-1894 (p < 0.001). All PRRSVisolates were significantly different (p < 0.001) from controls.

The precise mechanisms important in pathogenesis of PRRSV infection havenot been fully delineated. However, alveolar macrophages and epithelialcells lining bronchioli and alveolar ducts have been shown to containviral antigen by immunocytochemistry on frozen sections (Pol et al:Pathological, ultrastructural, and immunohistochemical changes caused byLelystad virus in experimentally induced infections of mystery swinedisease (synonym: porcine epidermic abortion and respiratory syndrome(PEARS). Veterinary Quarterly, 13:137-143 (1991)).

The present immunocytochemistry test for the detection of PRRSV informalin-fixed tissues (see Experiment VI supra) shows that PRRSV alsoreplicates in alveolar epithelial cells and macrophages. The extent ofvirus replication and cell types infected by PRRSV isolates also appearsto vary (see Experiment X below).

The role of different genes in virulence and replication is notprecisely known. However, ORF's 4 and 5 appear to be importantdeterminants of in vivo virulence and in vitro replication in PRRSV.

The results of cloning and sequencing ORF's 5, 6 and 7 of PRRSV isolateVR 2385 (see Experiment I supra) show that ORF 5 encodes a membraneprotein (also see U.S. application Ser. No. 08/131,625). A comparison ofORF's 5-7 of VR 2385 with ORF's 5-7 of Lelystad virus shows that ORF 5is the least-conserved of the three proteins analyzed (see Table 2supra), thus indicating that ORF 5 may be important in determiningvirulence.

Based on Northern blot results, ORF 4 of lv isolate VR 2431 appears tohave a deletion in mRNA 4 (also see Experiment V of U.S. applicationSer. No. 08/131,625).

EXPERIMENTS IX-XI

PRRSV (ATCC VR 2386) was propagated in vitro in ATCC CRL 11171 cells bythe method disclosed in Experiment III of U.S. application Ser. No.08/131,625. The PRRSV isolate was biologically cloned by three rounds ofplaque purification on CRL 11171 cells and characterized. Theplaque-purified isolate (hereinafter “VR 2386pp”, which is equivalent toVR 2386, deposited at the ATCC, Rockville Md., on Oct. 29, 1992)replicated to about 10⁶-10⁷ TCID₅₀/ml at the 11th cell culture passagein CRL 11171 cells. Viral antigens were also detected in the cytoplasmof infected cells using convalescent PRRSV serum. VR 2386pp was shown tobe antigenically related to VR 2332 by IFA using polyclonal andmonoclonal antibodies to the nucleocapsid protein of VR 2332 (SDOW-17,obtained from Dr. David Benfield, South Dakota State University).

Several other virus isolates (VR 2429 (ISU-22), ISU-28, VR 2428(ISU-51), VR 2430 (ISU-55), ISU-79, ISU-984, ISU-1894, and VR 2431(ISU-3927)) were isolated and plaque-purified on CRL 11171 cell line.Virus replication in the CRL 11171 cell line varied among PRRSV isolates(see Table 3 below). Isolate VR 2385 and plaque-purified isolates VR2386pp, VR 2430 and ISU-79 replicated to 10⁶⁻⁷ TCID₅₀/ml, and thus, havea high replication (hr) phenotype. Other isolates, such as ISU-984,ISU-1894 replicated to a titer of 10⁴⁻⁵ TCID₅₀/ml, corresponding to amoderate replication (mr) phenotype. Isolates ISU-3927 and ISU-984replicated very poorly on CRL 11171 cell line and usually yielded atiter of 10³ TCID₅₀/ml, and thus have a low replication (lr) phenotype.

EXPERIMENT IX

The pathogenicity of several PRRSV isolates was compared incesarean-derived colostrum-deprived (CDCD) pigs to determine if therewas a correlation between in vitro replication and pathogenicity (alsosee Experiment V of application Ser. No. 08/131,625. Fourplaque-purified PRRSV isolates (VR 2386pp, VR 2429, ISU-984, and VR2431), and one non-plaque-purified isolate (VR 2385) were used toinoculate pigs. An uninoculated group and an uninfected cellculture-inoculated group served as controls. Two pigs from each groupwere killed at 3, 7, 10, and 21 DPI. Three pigs were killed at 28 and 36DPI. Biologically cloned PRRSV isolates VR 2386pp, VR 2429, and ISU-984induced severe respiratory disease in the 5 week-old CDCD pigs, whereasVR 2431 did not produce any significant disease. Gross lung lesionscores peaked at 10 DPI (see Table 4) and ranged from 10.5%consolidation (VR 2431) to 77% consolidation (VR 2385). Lesions wereresolved by 36 DPI.

Microscopic lesions included interstitial pneumonia, encephalitis, andmyocarditis (Table 3). The lv isolates also caused less severemyocarditis and encephalitis than the hv isolates.

In FIGS. 28(A)-(C), photographs of lungs from pigs inoculated with (A)culture fluid from uninfected cell line CRL 11171, (B) culture fluidsfrom cell line infected with lv isolate VR 2431, (C) or culture fluidsfrom cell line infected with hv isolate VR 2386pp. The lung in FIG.28(B) has very mild pneumonia, whereas lung in FIG. 28(C) has severeconsolidation.

EXPERIMENT X

An additional experiment was conducted using a larger number of pigs tofurther examine the pathogenicity of PRRSV isolates and to obtain morestatistically significant data. Results are shown in Table 5.Collectively, the results show that PRRSV isolates can be divided intotwo groups based on pneumopathogenicity. Isolates VR 2385, VR 2429,ISU-28, and ISU-79 have a high virulence (hv) phenotype and producesevere pneumonia. Isolates ISU-51, VR 2430, ISU-1894 and VR 2431 have alow virulence (lv) phenotype (Table 4) and produce low grade pneumonia.

PRRSV isolates also produce two types of microscopic lesions in lungs.The first type found generally in lv isolates is designated as PRRS-A,and is characterized by interstitial pneumonia with septal infiltrationwith mononuclear cells typical of PRRS (as described by Collins et al,Isolation of swine infertility and respiratory syndrome virus (isolateATCC VR-2332) in North America and experimental reproduction of thedisease in gnotobiotic pigs. J. Vet. Diagn. Invest., 4:117-126 (1992)).The second type of lesion, PRRS-B, is found in hv isolates and ischaracterized as proliferative interstitial pneumonia with marked typeII pneumocyte proliferation, alveolar exudation and syncytial cellformation, as described in U.S. application Ser. No. 08/131,625 and byHalbur et al, An overview of porcine viral respiratory disease. Proc.Central Veterinary Conference, pp. 50-59 (1993). Examples of PRRS-A andPRRS-B type lesions are shown in FIGS. 28(A)-(C), in which FIG. 28(A)shows a normal lung, FIG. 28(B) are the lesions produced by PRRSV typeA, and FIG. 28(C) shows the lesions produced by PRRSV type B.

The immunoperoxidase assay of Experiment V using monoclonal antibodiesto PRRSV was used to detect viral antigens in alveolar epithelial cellsand macrophages (see FIG. 29(A)). This test is now being routinely usedat the Iowa State University Veterinary Diagnostic Laboratory to detectPRRSV antigen in tissues.

In FIGS. 29(A)-(B), immunohistochemical staining with anti-PRRSVmonoclonal antibody of lung from a pig infected 9 days previously withVR 2385. A streptavidin-biotin complex (ABC) immunoperoxidase techniquecoupled with hematoxylin counterstaining were used. Positive stainingwithin the cytoplasm of macrophages and sloughed cells in the alveolarspaces is clearly shown in FIG. 29(A), and within cellular debris interminal airway lumina in FIG. 29(B).

EXPERIMENT XI

To determine if there was a correlation between biological phenotypesand genetic changes in PRRSV isolates, Northern blot analyses wereperformed on 6 PRRSV isolates.

Total intracellular RNA's from the VR 2386pp virus-infected CRL 11171cells were isolated by the guanidine isothiocyanate method, separated on1% glyoxal/DMSO agarose gel and blotted onto nylon membranes. A cDNAprobe was generated by PCR with a set of primers flanking the extreme 3′terminal region of the viral genome. The probe contained 3′ noncodingsequence and most of the ORF-7 sequence (see U.S. application Ser. No.08/131,625).

Northern blot hybridization revealed a nested set of 6 subgenomic mRNAspecies (FIG. 30). The size of VR 2386pp viral genomic RNA (14.7 kb) andthe six subgenomic mRNA's, mRNA 2 (3.3 kb), mRNA 3 (2.8 kb), mRNA 4 (2.3kb), mRNA 5 (1.9 kb), mRNA 6 (1.4 kb) and mRNA 7 (0.9 kb), resembledthose of LV, although there were slight differences in the estimatedsizes of the genome and subgenomic mRNA's (Conzelmann et al, Virology,193, 329-339 (1993), Meulenberg et al, Virology, 192, 62-72 (1993). ThemRNA 7 of the VR 2386pp was the most abundant subgenomic mRNA (see FIG.30 and Experiment I above). The total numbers of subgenomic mRNA's andtheir relative sizes were also compared. The subgenomic mRNA's of threeisolates had 6 subgenomic mRNA's, similar to that described for Lelystadvirus. In contrast, three isolates had 8 subgenomic mRNA's (FIG. 30).The exact origin of the two additional species of mRNA's is not known,but they are located between subgenomic mRNA's 3 and 6 and were observedrepeatedly in cultures infected at low MOI. Interestingly, an additionalsubgenomic mRNA has been detected in LDV isolates propagated inmacrophage cultures (Kuo et al, 1992). We speculate that the additionalmRNA's in cells infected with some PRRSV isolates are derived from gene4 and 5 possibly transcribed from an alternate transcriptional startsite. Additional studies are needed to determine the origin of theseRNA's and their significance in pathogenesis of PRRSV infections.

FIG. 30 shows Northern blots of PRRSV isolates VR 2386pp (designated as“12”), VR 2429 (ISU-22, designated as “22”), VR 2430, designated as“55”), ISU-79 (designated as “79”), ISU-1894 (designated as “1894”), andVR 2431, designated as “3927”). This data represents results from fourseparate Northern blot hybridization experiments. The VR 2386pp isolate(12) was run in one gel, ISU-1894 and VR 2431 were run in a second gel,VR 2430 and ISU-79 were run in a third gel, and ISU-22 was run in afourth gel. Two additional mRNA's are evident in isolates VR 2429, VR2430, and ISU-79.

The subgenomic mRNA 4 of VR 2431 (ISU-3927) migrates faster than that ofother isolates in Northern blotting, suggesting a deletion.Interestingly, the isolate VR 2431 has lv and lr phenotypes and is theleast virulent PRRSV isolate of the Iowa strains described herein. Thissuggests that gene 4 may be important in virulence and replication. Asdescribed above, genes 6-and 7 are less likely to play a role inexpression of virulence and replication phenotypes.

In summary, PRRSV isolates vary in pathogenicity and the extent ofreplication in cell cultures. The number of subgenomic mRNA's and theamount of mRNA's also varies among U.S. PRRSV isolates. Moresignificantly, one of the isolates, VR 2431, which replicates to lowtiter (lr phenotype) and which is the least virulent isolate (lvphenotype) among the Iowa strain PRRSV isolates described herein,appears to have a faster migrating subgenomic mRNA 4, thus suggestingthat a deletion exists in its ORF 4.

EXPERIMENT XII Comparison of the Pathogenticity and Antigen Distributionof Two U.S. Porcine Reproductive and Respiratory Syndrome Virus Isolateswith the Lelystad Virus

PRRSV-induced respiratory disease with secondary bacterial pneumonia,septicemia and enteritis are frequently observed in 2-10-week-old pigs(Halbur et al., “Viral contributions to the porcine respiratory diseasecomplex,” Proc. Am. Assoc. Swine Pract., pp. 343-350 (1993); Zeman etal., J. Vet. Diagn. Invest. (1993)). Outbreaks may last from 1-4 monthsor become an ongoing problem on some farms where pig-flow through theunit is appropriate for shedding of the virus from older stock toyounger susceptible animals that have lost passive antibody protection.

The severity and duration of outbreaks is quite variable. In fact, someherds are devastated by the high production losses (Polson et al.,“Financial Impact of Porcine Epidemic Abortion and Respiratory Syndrome(PEARS),” Proc. 12th Inter. Pig Vet. Soc., p. 132 (1992); Polson et al,“An evaluation of the financial impact of porcine reproductive andrespiratory syndrome (PRRS) in nursery pigs,” Proc. 13th Inter. Pig Vet.Soc., p. 436 (1994)), while other herds have no apparent losses due toinfection with PRRSV. This may be due to a number of possibilities,including virus strain differences, pig genetic susceptibilitydifferences, environmental or housing differences, or production style(pig flow) of the unit.

This experiment compares the pathogenicity and antigen distribution oftwo U.S. strains (ISU-12 [VR 2385], ISU-3927 [VR 2431]) and a Europeanstrain (Lelystad virus, obtained from the National Veterinary ServicesLaboratory, P.O. Box 844, Ames, Iowa, 50010) in a common pig model todocument similarities and differences that may explain the differencesin severity of field outbreaks of PRRSV and help to better understandthe pathogenesis of disease induced by PRRSV. (In the followingexperimental descriptions, “x/y” refers to the number of pigs “x” out ofa particular group of pigs having “y” members.)

Materials and Methods

Experimental Design:

One hundred caesarian-derived-colostrum-deprived (CDCD) pigs of 4 weeksof age were randomly divided into 4 large groups of 25 pigs each andassigned to one of four isolated buildings. Within each building, thepigs were further divided into 3 separate rooms (11 pigs, 11 pigs, and 3pigs per room). Each room within the buildings had separate, automatedventilation systems. The pigs were housed on raised woven wire decks andfed a complete 18% protein corn and soybean meal based ration. Followingchallenge with a virus inoculum, the pigs were necropsied as detailed inTable 6 below at 1, 2, 3, 5, 7, 10, 15, 21 and 28 days post inoculation(DPI).

Virus Inocula Preparation:

Each virus was plaque-purified three times. Challenge doses were10^(5.8) for VR 2385 and 10^(5.8) for VR 2431. The challenge dose ofLelystad virus was 10^(5.8).

Pigs were challenged intranasally by sitting them on their buttocksperpendicular to the floor and extending their neck fully back. Theinocula was slowly dripped into both nostrils of the pigs, takingapproximately 2-3 minutes per pig. Control pigs were given 5 mL ofuninfected cell culture media in the same manner.

Clinical Evaluation

Rectal temperatures were taken and recorded daily from −2 DPI through 10DPI. A clinical respiratory disease score was given to each pig dailyfrom day 0 to 10 DPI, in accordance with the following 0-6 score range,similar to the respiratory distress analysis described above:0=normal

TABLE 6 Necropsy Schedule 1 2 3 5 7 10 15 21 28 Isolate Room DPI DPI DPIDPI DPI DPI DPI DPI DPI Total Lelystad 1 1 1 1 1 1 3 1 1 1 11 Lelystad 21 1 1 1 1 3 1 1 1 11 Lelystad 3 3 3 VR 2385 4 1 1 1 1 1 3 1 1 1 11 VR2385 5 1 1 1 1 1 3 1 1 1 11 VR 2385 6 3 3 Control 7 1 1 1 1 1 3 1 1 1 11Control 8 1 1 1 1 1 3 1 1 1 11 Control 9 3 3 VR 2431 10 1 1 1 1 1 3 1 11 11 VR 2431 11 1 1 1 1 1 3 1 1 1 11 VR 2431 12 3 31 = mild dyspnea and/or tachypnea when stressed2 = mild dyspnea and/or tachypnea when not stressed3 = moderate dyspnea and/or tachypnea when stressed4 = moderate dyspnea and/or tachypnea when not stressed5 = severe dyspnea and/or tachypnea when stressed6 = severe dyspnea and/or tachypnea when not stressed

A pig was considered “stressed” by the pig handler after holding the pigunder his/her arm and taking the pig's rectal temperature forapproximately 30-60 seconds. Other relevant clinical observations likecoughing, diarrhea, inappetence or lethargy were noted separately, andare not reflected in the respiratory disease score.

Pathologic Examination:

Complete necropsies were performed on all pigs. Macroscopic lung lesionswere given a score to estimate the percent consolidation of the lung.Each lung lobe was assigned a number to reflect the approximate volumeof entire lung represented by that lobe. Ten (10) possible points wereassigned to each of the right anterior lobe, right middle lobe, anteriorpart of the left anterior lobe, and caudal part of the left anteriorlobe of the lung. The accessory lobe was assigned five (5) points.Twenty-seven and one-half (27.5) points were assigned to each of theright and left caudal lobes to reach a total of 100 points. Gross lunglesion scores were estimated, and a score was given to reflect theamount of consolidation in each lobe. The total for all the lobes was anestimate of the percent consolidation of the entire lung for each pig.

Sections were taken from all lung lobes, nasal turbinates, cerebrum,thalamus, hypothalamus, pituitary gland, brain stem, choroid plexus,cerebellum, heart, pancreas, ileum, tonsil, mediastinal lymph node,middle iliac lymph node, mesenteric lymph node, thymus, liver, kidney,and adrenal gland for histopathologic examination. Tissues were fixed in10% neutral-buffered formalin for 1-7 days and routinely processed toparaffin blocks in an automated tissue processor. Sections were cut at 6μm and stained with hematoxylin and eosin.

Immunohistochemistry:

Immunohistochemical staining was performed as described in ExperimentVI-above. Sections were cut at 3 μm and mounted on poly-L-lysine coatedslides. Endogenous peroxidase was removed by three 10-minute changes of3% hydrogen peroxide. This was followed by a TRIS bath, and thendigestion with 0.05% protease (Protease XIV, Sigma Chemical Company, St.Louis, Mo.) in TRIS buffer for 2 minutes at 37° C. After another TRISbuffer bath, blocking was done for 20 minutes with a 5% solution ofnormal goat serum. Primary monoclonal antibody ascites fluid (SDOW-17,obtained from Dr. David Benfield, South Dakota State Univ.) diluted1:1000 in TRIS/PBS was added for 16 hours at 4° C. in a humidifiedchamber. After primary antibody incubation and a subsequent 5 minuteTRIS bath containing 1% normal goat serum, the slides were flooded withbiotinylated goat anti-mouse linking antibody (Dako Corporation,Carpintera, Calif.) for 30 minutes. The sections were washed with TRISand treated with peroxidase-conjugated streptavidin (Zymed Laboratories,South San Francisco, Calif.) for 40 minutes, then incubated with3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories Inc.,Burlingame, Calif.) for 8-10 minutes. Sections were then stained withhematoxylin.

Immunohistochemical controls substituted TBS for the primary antibody onall lung and lymphoid tissue sections. The same was done on othersections of other tissues interpreted as possibly positive. Uninfectedcontrol pigs also served as negative controls. No staining was detectedin any of the control pig tissues. The-amount of antigen was estimatedaccording to the following scale: (0)=negative (no positive cells),(1)=isolated or rare positive staining cells (about 1-5 positive cellsper histologic section), (2)=a relatively low number of positive cells,yet more abundant than isolated cells (for example, about 10-20 positivecells per histologic section), (3)=a moderate number of positive cells(for example, about 40-80 positive cells per histologic section), and(4)=a relatively large number of positive cells (more than about 100positive cells per histologic section).

Virus Isolation:

The same tissues from each of two pigs necropsied from each challengegroup were pooled at 1, 2, 3, 5, 7, 14, 21, and 28 DPI. At 10 DPI, ninepigs were necropsied from each group, so three pools of the same tissuesfrom three pigs were made from each challenge group. Serum was alsosimilarly pooled.

Results

Clinical Disease:

The mean clinical respiratory disease score for each group is summarizedin Table 7. Control pigs remained normal. Respiratory disease wasminimal, and symptoms and histopathology were similar in the groups ofpigs infected with Lelystad virus and VR 2431. By 2 DPI, a few pigs ineach of these groups demonstrated mild dyspnea and tachypnea after beingstressed by handling. From 5-10 DPI, more of the pigs in these groupsdemonstrated mild respiratory disease, and a couple pigs evidencedmoderate, but transient, labored abdominal respiration. By 14 DPI, TABLE7 Mean Clinical Respiratory Disease Score 0 1 2 3 4 5 6 7 8 9 10 GROUPDPI DPI DPI DPI DPI DPI DPI DPI DPI DPI DPI Control 0 0 0 0 0 0 0 0.1 00.1 0 Lelystad 0 0.2 0.1 0.2 0.5 0.6 0.8 1.0 0.9 0.3 0.3 VR 2431 0 0 0.30.2 0.4 0.6 0.3 1.3 0.7 0.5 0.5 VR 2385 0 0.4 1.5 1.8 2.2 3.2 3.4 3.53.3 3.4 3.0all pigs in the Lelystad virus (LV) and VR 2431 groups had recovered.Other transient clinical disease noted in a few pigs in these groupsincluded chemosis, reddened conjunctiva, ear drooping, and patchycyanosis of skin when stressed by handling. Coughing was not observed.

By 2 DPI, the VR 2385-challenged group demonstrated mild respiratorydisease without having been stressed. By 5 DPI, all of the pigs in thisgroup demonstrated moderate respiratory disease characterized by laboredabdominal respiration and dyspnea when stressed. Some of the pigs inthis group received respiratory distress scores of 5 or 6 for a 2- to5-day period, and the mean clinical respiratory disease score peaked at3.5/6 at 7 DPI. Respiratory disease was characterized by severetachypnea and labored abdominal respiration, but no coughing wasobserved. The VR 2385 pigs generally were moderately lethargic andanorexic from 4-10 DPI. Other transient clinical signs includedchemosis, roughed hair coats, lethargy, and anorexia. It took up to 21DPI for the majority of the pigs in this group to fully recover.

Gross Lesions

Table 8 summarizes the estimated percent consolidation of the lungs forpigs in each group. Lung lesions in the Lelystad group and VR 2431 groupwere similar in type and extent. Lesions were first observed at 5 DPIfor both groups, and peaked at 15 DPI for the Lelystad challenged groupand at 7 DPI for VR 2431 challenged group. Individual scores ranged from0-31 percent consolidation for the Lelystad group and 0-27 percent forthe VR 2431 group. The mean estimated percent consolidation of the lungfor the nine pigs necropsied at 10 DPI was 6.8 percent for Lelystadvirus challenged pigs and 9.7 percent for the VR 2431 challenged pigs.The lesions were predominately in the cranial, middle and accessorylobes and in the ventromedial portion of the diaphragmatic lobes. Theconsolidation was characterized by multifocal, tan-mottled areas withirregular, indistinct borders. TABLE 8 Estimated Percent Consolidationof Lungs (0-100%) 1 2 3 5 7 10 15 21 28 GROUP DPI DPI DPI DPI DPI DPIDPI DPI DPI Con- 0 0 0 0 0 0 0 0 0 trol Lely- 0 0 0 4.8 2.3 6.8 8.8 1.80 stad VR 0 0 0 2.5 8.5 9.7 7.5 0 0 2431 VR 0 4.3 10.5 15.3 46.5 54.212.5 6.0 0 2385

Gross lymphoid lesions were more common than lung lesions with both VR2431 and LV. Lymphadenopathy was consistently observed in themediastinal and middle iliac lymph nodes. These lymph nodes were tan incolor, and from 5-28 DPI, were enlarged to 2-10 times their normal size.There often was at least one 1-5 mm fluid-filled cyst in each of theselymph nodes. No other gross lesions were observed in the LV or VR 2431groups.

The VR 2385 group had considerably more severe lung consolidation. Thedistribution of lung consolidation was similar to pigs infected with VR2431 and LV, but either the entire cranioventral lobes or largecoalescing portions of the cranial, middle, accessory and ventromedialdiaphragmatic lobes were consolidated. There was no pleuritis and nogrossly visible pus in airways. Estimated percent consolidation of thelung 7-10 DPI ranged from 28% to 71%. The estimated mean score of thenine pigs necropsied at 10 DPI was 54.2% consolidation.

Lymphoid lesions in the VR 2385 group were generally similar to thoseobserved in the other groups. Additionally, lymph nodes along thethoracic aorta and in the cervical region were often 2-5 times thenormal size. Spleens were also slightly enlarged and meaty in texture.

Several pigs in the VR 2385 group had moderately enlarged and roundedhearts with 10-30 mL of clear fluid in the pericardial space. Some ofthese pigs also had 50-200 mL of similar fluid in the abdominal cavity.There was no visible exudate or fibrin in the fluid.

Microscopic Lesions:

Heart: Control pigs necropsied up to 10 DPI had no evidence ofmyocardial inflammation. Several pigs throughout the study had randomlydistributed discrete foci of hematopoietic cells in the endocardium andmyocardium. These hematopoietic cells (i) were observed in clumps of10-30 cells, (ii) ranged in size from 8-20 microns, and (iii) had largeround-oval, dark staining nuclei with dense, clumped chromatin, multiplesmall nucleoli and scant amphophilic cytoplasm. At 10 DPI, 2/9 controlpigs had mild multifocal perivascular lymphohistiocytic myocarditis.This was also observed in 1/2 pigs necropsied at 15 and 21 DPI,respectively.

VR 2431 inoculated pigs also had evidence of myocardial extramedullaryhematopoiesis, similar to the controls. Myocarditis was first observedat 7 DPI, and was seen in 16/18 pigs necropsied from 7-28 DPI. Themyocarditis was mild, multifocal, usually perivascular and peripurkinje,and lymphohistiocytic. Inflammation was consistently found in theendocardium, often around or involving purkinje fibers. Inflammation inthe epicardium and myocardium was most consistently either aroundvessels or randomly distributed between muscle fibers. Myocardialdegeneration, necrosis, or fibrosis was not evident. Low numbers ofeosinophils were observed in the perivascular infiltrates in a 4/9 pigsat 9 DPI.

In the LV inoculated pigs, mild multifocal extramedullary hematopoiesiswas evident in most pigs up to 7 DPI. Mild myocarditis was firstobserved at 2 DPI and was inconsistent and mild in pigs posted from 3-10DPI. The pigs necropsied at 15 and 21 DPI had moderate multifocalmyocarditis. The myocarditis was much less severe by 28 DPI. In all,13/17 LV inoculated pigs necropsied from 7-28 DPI had lymphohistiocyticmyocarditis, which was mild-moderate, perivascular, peripurkinje orrandom in distribution. Fewer numbers of plasma cells and eosinophilswere found in areas of inflammation from 10-28 DPI.

Moderate multifocal lymphohistiocytic myocarditis was observed beginningat 10 DPI in all of the VR 2385 inoculated pigs. Severe myocarditis wasobserved in 2/9 pigs killed at 10 DPI and in 1/2 pigs killed at each of15, 21, and 28 DPI, respectively. The more severe cases werecharacterized by multifocal-to-diffuse, lymphoplasmacytic andhistiocytic infiltrates that were most intense in the perivascular,peripurkinje, and endocardial regions. Lesser numbers of eosinophils andunidentifiable pyknotic cells were also observed in association with theinflammation. Myocardial degeneration, necrosis and fibrosis were notevident.

Lung: Very mild lung lesions were observed in 2/25 of the control pigs.One pig necropsied at 5 DPI had mild multifocal septal thickening withlymphocytes, macrophages, and neutrophils. At 10 DPI, one pig had mildperibronchiolar and perivascular lymphohistiocytic cuffing and a mildincreased number of macrophages and neutrophils in the alveolar spaces.

In the VR 2431 inoculated pigs, microscopic lung lesions were firstdetected at 2 DPI and were present in 20/25 of the pigs. All pigsnecropsied on or after 7 DPI had microscopic lung lesions. The lesions,when present, were multifocal, mild (12/25) to moderate (8/25),generally most severe at 10 DPI and nearly resolved at 28 DPI. Themultifocal interstitial pneumonia was characterized by three primarychanges: septal thickening with mononuclear cells, type 2 pneumocytehypertrophy and hyperplasia, and accumulation of normal and necroticmacrophages in alveolar spaces. These changes were present throughoutthe 28-day period. Mild-to-moderate peribronchiolar and perivascularlymphohistiocytic cuffing was observed in most pigs examined at 10-15DPI but had apparently resolved by 28 DPI. Lung lesions were seldomobserved in sections taken from the caudal lung lobe.

The LV inoculated pigs had microscopic lung lesions very similar tothose of VR 2431 in distribution, type, and severity. Microscopic lunglesions were observed in 21/25 of the LV pigs. Lesions were firstobserved at 2 DPI and persisted throughout the 28 day period. The mostsevere lesions were seen in a few of the pigs necropsied at 10 DPI andin most of those necropsied at 15 and 21-DPI. The interstitial pneumoniawas characterized mainly by septal thickening with mononuclear cells,peribronchiolar and perivascular lymphohistiocytic cuffing, andaccumulation of macrophages and necrotic debris in alveolar spaces. Type2 pneumocyte hyperplasia and hypertrophy was less consistent and lesssevere than that observed in the VR 2431 inoculated pigs. Lung lesionswere seldom seen in sections taken from the caudal lung lobe.

Every pig that was inoculated with VR 2385 and necropsied on or after 5DPI had moderate-to-severe interstitial pneumonia. Mild multifocallesions were observed at 2 DPI. The lesions became moderate andmultifocal by 5 DPI, severe and diffuse from 7-10 DPI, and stillmoderate but patchy at 21 and 28 DPI. The interstitial pneumonia at allstages was also characterized by three primary changes (septalthickening with mononuclear cells, type 2 pneumocyte hypertrophy andhyperplasia, and accumulation of normal and necrotic macrophages inalveolar spaces). Of these three changes, the pneumocyte hypertrophy wasmost prominent and characteristic of VR 2385 inoculation.Peribronchiolar and perivascular lymphomacrophagic cuffing was mild by 5DPI, moderate by 10 DPI, and nearly resolved by 28 DPI.

Immunohistochemistry

Both adrenal glands were examined from all pigs. Adrenal gland lesionswere not observed in any of the control, VR 2431 or LV inoculated pigs.In the VR 2385 inoculated pigs, 9/25 pigs had mild multifocallymphoplasmacytic and histiocytic adrenalitis. Inflammation was usuallyobserved in the medulla. Pyknotic cells and karryhectic debris were alsoobserved amongst the inflammatory cells. Lymphoplasmacytic vasculitisand neuritis were also observed in the adrenal artery and nerve,respectively, in 3/28 of the VR 2385 inoculated pigs.

Nasal turbinate lesions were similar in type but differed in severityand frequency in the 4 groups of pigs. A low number (5/25) of thecontrol and LV (5/25) inoculated pigs had mild rhinitis, observed at10-21 DPI. The rhinitis was characterized by patchy dysplasia of theepithelium, with loss of cilia and mild multifocal subepitheliallymphohistiocytic and suppurative inflammation, with slight edema andcongestion.

More of the VR 2431 inoculated pigs (17/25) had rhinitis. Lesions weremild at 5 DPI but moderate by 10 DPI. Epithelial dysplasia withintercellular edema, a blebbed or “tombstone” appearance of swollensuperficial epithelial cells becoming pyknotic and apparently sloughinginto the nasal cavity, and complete or partial loss of cilia on largepatches of epithelium were observed. There was moderate diffusesubepithelial edema, dilated and congested veins, and multifocalinfiltrates of lymphocytes, plasma cells, macrophages and neutrophils.The inflammation was most intense near the locations where the ducts ofsubmucosal mucous glands extended to the surface. Leukocytic exocytosis,especially of neutrophils, were frequently observed in dysplasticsurface epithelium and along mucous ducts. By 21 DPI, the lesions hadbecome mild, and were resolved by 28 DPI.

Rhinitis was first observed at 5 DPI in the VR 2385 inoculated pigs. Atotal of 20/25 pigs, and all 17 pigs necropsied on or after 7 DPI, hadrhinitis similar to that observed in the ISU-3927 group, except that thelesion persisted throughout the 28 day period.

Tables 9, 10, and 11 summarize and compare the number of differenttissues in which PRRSV antigen was detected for each of the challengegroups. No antigen was detected in the control pigs. Table 12 summarizesthe estimated amount of antigen in some of the tissues that were tested.

Virus Isolation

Virus isolation from various tissues is summarized in Table 13, where“Lg” refers to lungs, “LN” refers to lymph nodes, “Ht” refers to theheart, “Ser” refers to serum, “Tons” refers to tonsils, “Spln” refers tothe spleen, “SI” refers to small intestine, and “Brn” refers to thebrain. TABLE 9 Immunohistochemistry for VR 2385 1 2 3 5 7 10 15 21 28Tissue DPI DPI DPI DPI DPI DPI DPI DPI DPI Total Lung 0/2 1/2 2/2 2/22/2 9/9 2/2 2/2 2/2 22/25 TBLN 1/2 2/2 2/2 2/2 2/2 3/9 0/2 1/2 0/2 13/25Med LN 0/2 2/2 2/2 2/2 2/2 4/9 0/2 0/2 2/2 14/25 Iliac LN 1/2 2/2 2/22/2 2/2 5/9 0/2 0/2 0/2 14/25 Tonsil 2/2 2/2 2/2 2/2 2/2 9/9 2/2 2/2 2/225/25 Thymus 0/2 1/2 2/2 2/2 2/2 2/9 0/2 0/2 0/2  9/25 Spleen 0/2 2/22/2 2/2 0/2 3/9 0/2 1/2 0/2 10/25 # pos 2/2 2/2 2/2 2/2 2/2 9/9 2/2 2/22/2 25/25

TABLE 10 Immunohistochemistry for VR 2431 1 2 3 5 7 10 15 21 28 TissueDPI DPI DPI DPI DPI DPI DPI DPI DPI Total Lung 1/2 1/2 0/2 1/2 0/2 7/92/2 0/2 2/2 14/25 TBLN 0/2 2/2 1/2 2/2 2/2 1/9 0/2 0/2 0/2  8/25 Med LN0/2 2/2 2/2 2/2 1/2 1/9 0/2 0/2 2/2 10/25 Iliac LN 0/2 2/2 2/2 2/2 1/21/9 0/2 0/2 0/2  8/25 Tonsil 1/2 1/2 1/2 2/2 1/2 9/9 2/2 2/2 2/2 21/25Thymus 0/2 0/2 2/2 1/2 1/2 0/9 0/2 2/2 0/2  6/25 Spleen 0/2 0/2 0/2 0/20/2 0/9 0/2 0/2 1/2  1/25 # pos 1/2 2/2 2/2 2/2 2/2 9/9 2/2 2/2 2/225/25

TABLE 11 Immunohistochemistry for Lelystad virus 1 2 3 5 7 10 15 21 28Tissue DPI DPI DPI DPI DPI DPI DPI DPI DPI Total Lung 0/2 1/2 1/2 1/21/2 5/9 2/2 2/2 1/2 14/25 TBLN 1/2 1/2 1/2 0/2 1/2 5/9 0/2 0/2 0/2  9/25Med LN 1/2 1/2 2/2 1/2 1/2 2/9 0/2 1/2 1/2 10/25 Iliac LN 0/2 1/2 2/20/2 1/2 0/9 0/2 0/2 0/2  4/25 Tonsil 2/2 2/2 2/2 2/2 2/2 7/9 2/2 2/2 2/223/25 Thymus 0/2 0/2 0/2 2/2 0/2 0/9 0/2 0/2 0/2  2/25 Spleen 1/2 1/20/2 0/2 0/2 4/9 0/2 0/2 1/2  7/25 # pos 2/2 2/2 2/2 2/2 2/2 8/9 2/2 2/22/2 25/25Serology

All pigs challenged with LV virus were negative prechallenge andremained <1:20 through 7 DPI. By 10 DPI, 6/9 of the pigs necropsied wereseropositive with titers ranging from 1:20 to 1:1280. Only 2/10 pigs hadtiters >1:20 (both were 1:1280). By 15 DPI, all pigs were positive and5/6 were >1:320. By 21 DPI, titers of 1:1280 or 1:5120 were most common.The VR 2431 antibody titers were similar to those levels seen with theLV virus. With VR 2385, however, 9/9 were positive by 10 DPI and 7/9were ≧1:320. No PRRSV serum antibody was detected in control pigs.

Discussion

This Experiment clearly demonstrates differences in pathogenicitybetween PRRSV isolates, differences in PRRSV antigen distribution, anddifferences in the amount of PRRSV antigen in selected tissues. The lowvirulence Iowa strain isolate VR 2431 and the low virulence Lelystadvirus were similar in these criteria. The Iowa strain VR 2385 isolatewas considerably more virulent, and PRRSV antigen was detected in moretissues and in greater amounts as compared to LV and VR 2431.

The pattern of antigen distribution over time (Table 12) suggests thatwhen pigs are infected oronasally, initial and continual replication ofthe virus may be in TABLE 12 Mean score for intensity/amount of PRRSVantigen detected by immunohistochemistry VR 2385 VR 2431 Lelystad VirusCrVn Mid Med lliac CrVn Mid Med lliac CrVn Mid Med lliac DPI Lung LungTBLN LN LN Tonsil Lung Lung TBLN LN LN Tonsil lung Lung TBLN LN LNTonsil 1 0 0 1.5 0 0.5 1.0 0.5 0 0 0 0 0.5 0 0 0.5 0.5 0 1.0 2 0.5 1.02.0 1.5 2.0 1.5 0.5 0 2.0 1.0 2.5 0.5 0.5 0.5 1.0 1.0 1.0 1.0 3 2.0 2.53.0 3.0 3.0 3.0 0 0 1.0 1.5 2.5 0.5 0.5 0.5 1.0 1.5 2.0 1.0 5 2.0 2.03.0 3.0 2.5 3.0 0.5 1 2.0 2.0 2.0 1.0 1.0 0.5 0 0.5 0 1.0 7 2.5 1.5 1.01.5 2.0 1.0 0 2 1.0 1.0 0.5 0.5 1.0 0 1.0 0.5 0.5 1.0 10 2.0 1.6 0.5 0.60.7 1.2 1.1 0.9 0.1 0.1 0.1 1.1 0.3 0.4 0.6 0.2 0 0.8 15 1.0 0 0 0 0 1.02.0 0.5 0 0 0 1.0 0.5 0.5 0 0 0 1.0 21 2.0 0.5 0.5 0 0 2.5 0 0 0 0 0 1.01.0 0 0 0.5 0 1.5 28 1.0 0 0 1 0 1.5 1.3 0 0 1.3 0 2.0 0.5 0 0 0.5 0 1.0Antigen amount was estimated and scored as follows: (0) = negative, (1)= isolated or rare positive staining cells, (2) = low number of positivecells, (3) moderate number of positive cells, and (4) = large number ofpositive cells.CrVn = Cranioventral lung lobe;Mid = middle lung lobe;TBLN = tracheobronchial lymph node;Med LN = mediastinal lymph node.

TABLE 13 Virus isolation VR 2385 VR 2431 Lelystad Virus DPI Lg LN Ht SerTons Spln SI Brn Lg LN Ht Ser Tons Spln SI Brn Lg LN Ht Ser Tons Spln SIBr 1 + + − + − − + − + − − + + − + − − − − + + − − −2 + + + + + + + + + + + + + + + − + + + + + + + −3 + + + + + + + + + + + + + + + − + + + + + + + −5 + + + + + + + + + + + + + + − + + + − + + + + −7 + + + + + + + + + + + + + + − + + + + + + + + − 10 + + + + + + +− + + + + + + − + + + + + + + + − 10 + + − + + + + − + + + + + + −− + + + + + + + − 10 + + + + + + + + + − + + + + − + + + + + + + − +15 + + + + + + + − + + + + + + − − + + + + + + − − 21 + + + + + + + − +− − + + − − − + − + + + − − − 28 + + − + + − − − + − + + + − − − +− + + + − − −the tonsil and upper respiratory tract lymphoid tissues, with subsequentviremia by 24 hours PI. A small amount of antigen is detected in thelung by 24 hours PI and peaks by 5-7 DPI, but persists there for up to28 days. Antigen is present in lymphoid tissues generally from 2-21 DPI.

Antigen is detected primarily within the macrophages and dendritic-likecells in lung, lymph nodes, tonsil, thymus and spleen.

EXPERIMENT XIII Comparative Pathogenicity of Nine U.S. PRRSV Isolates ina 5 Week Old CDCD Pig Model

Part (A) of this experiment demonstrates a consistent model to studyPRRSV-induced respiratory and systemic disease in piglets (e.g., about 5weeks old) and to characterize gross and microscopic lesions associatedwith the course of PRRSV-induced disease. Part (B) of this experimentuses the model to statistically compare the virulence of PRRSV isolatesfrom herds with differing disease severity, and to specificallydetermine if these differences may be due to virus virulencecharacteristics.

Materials and Methods

Source of PRRSV Isolates:

Live pigs or fresh tissues were received from 61 herds over a 3-yearperiod from 1991-1993. All cases were submitted for etiologic diagnosisof respiratory disease in pigs from 1-16 weeks of age. Some of the herdshad concurrent reproductive failure, and some did not. The nine selectedherds differed in size, production style, age 5 of diseased pigs, timesince initial disease was observed, and severity of the current diseaseoutbreak. The clinical information from the selected farms is summarizedin Table 14. TABLE 14 PRRSV Herd Profiles Production Age of Type ofIsolate Herd Size Style Disease Disease VR 2385 180 sows F-Fin/CF ALLsevere PRRS ISU-79 40 sows F-Fin/AIAO ALL severe PRRS ISU-28 150 sowsF-Fin/CF ALL severe PRRS ISU-1894 600 sows F-FRP/CF 3-8 weeks severeresp. VR 2428 900 sows F-FRP/AIAO 3-8 weeks severe resp. VR 2429 100sows F-Fin/CF 1-8 weeks moderate resp. ISU-984 600 sows F-FRP/AIAO 3-6weeks moderate resp. VR 2430 150 sows F-Fin/CF 3-6 weeks mild resp. VR2431 60 sows F-Fin/AIAO 1-4 weeks mild resp.F-Fin = Farrow-to-FinishF-FRP = Farrow-to-Feeder PigCF = Continuous FlowAIAO = All-in-All-outInocula Preparation

PRRSV isolates were plaque purified 3 times in accordance with theprocedure described in Experiment I, section (I)(A) above.

Experimental Pigs:

Four-week-old caesarean-derived-colostrum-deprived (CDCD) pigs wereinitially fed a commercial 22% protein pig starter containingspray-dried plasma protein for 7 days, then were switched to a secondstage 18% protein corn-soybean meal based ration for the duration of theexperiment. Pigs were housed in 10 feet×12 feet concrete-floored,individually power-ventilated rooms.

Part (A): CDCD Pig Model:

Ninety-eight 4-week-old CDCD pigs were randomly divided into-7 rooms of14 pigs each. The rooms were randomly assigned-one of seven treatmentsas shown in Table 15. The treatment consisted of intranasal inoculationof 10^(5.7) TCID₅₀ of a PRRSV isolate (selected from plaque-purifiedPRRSV isolates VR 2385, VR 2428 [ISU-22], VR 2431 or ISU-984,unplaque-purified isolate ISU-12 [VR 2386]), intranasal inoculation ofuninfected cell culture and media, or no treatment. Two pigs from eachgroup were necropsied at DPI 3, 7, 20 and 21, and 3 pigs were necropsiedfrom each group at DPI 28 and 36. Rectal temperatures were recordeddaily from DPI −2 though DPI +14. A clinical respiratory disease scorewas given from DPI −2 through DPI 14. Scores range from 0-6, inaccordance with the respiratory distress scale recited in ExperimentXII. A piglet was considered “stressed” by the pig handler when holdingthe pig under his/her arm and taking the rectal temperature forapproximately 30-60 seconds. Other relevant clinical observations (e.g.,coughing, diarrhea, inappetence or lethargy) were noted separately asobserved. Additional clinical observations had no impact on the clinicalrespiratory score. Weights were recorded an DPI 0, 7, 14, 21 and 28.TABLE 15 Part (A) Experimental Design 3 7 10 21 28 36 Total Inoculum DPIDPI DPI DPI DPI DPI Pigs VR 2385 2 2 2 2 3 3 14 ISU-984 2 2 2 2 3 3 14VR 2428 2 2 2 2 3 3 14 VR 2431 2 2 2 2 3 3 14 VR 2386 2 2 2 2 3 3 14Uninoculated Control 2 2 2 2 3 3 14 PSP-36 Cell Culture 2 2 2 2 3 3 14Part (B): Comparative Pathogenicity:

Results from Part (A) established that gross lung lesions were mostsevere at 10 DPI for 4 of 5 PRRSV isolates. Part (B) was designed tocollect and compare data from a larger number of pigs necropsied at 10DPI. In this experiment, 105 4-week-old crossbred CDCD pigs wererandomly divided into seven rooms, each with 15 pigs. Each room wasrandomly assigned a treatment. Treatments consisted of intranasalchallenge with 10^(5.8) TCID₅₀ of one of six plaque-purified PRRSVisolates (VR 2429 [ISU-51], ISU-79, VR 2430 [ISU-55], ISU-1894, ISU-28or VR 2385) or PSP-36 uninfected cell culture and media. Ten pigs fromeach group were necropsied at 10 DPI, and 5 pigs from each group werenecropsied at 28 DPI. Rectal temperatures were recorded from −2 DPI to+10 DPI, and weights were recorded at 0, 10 and 28 DPI. Clinicalrespiratory disease scores and other clinical signs were recorded as inPart (A) above.

Serology:

Part (A): Pigs were bled at 0, 10 and 28 DPI. The presence of PRRSVserum antibody was detected by the immunofluorescent antibody technique(IFA) as described by Benfield et al (J. Vet. Diagn. Invest., 4:127-133(1992)).

Part (B): Pigs were bled at 0, 3, 10, 16 and 28 DPI and tested by theIFA procedure of Part (A) for the presence of PRRSV serum antibody.

Virus Isolation:

Virus isolation was attempted from lung homogenates of all pigs killedat 3, 7, 10, 21 and 28 DPI (Part (A)). Virus isolation was alsoattempted from lung and from serum of all pigs separately in two-pigpools using CRL 11171 (PSP 36) cells (Part (B)).

Gross Pathology:

Complete necropsies were performed on all pigs. All organ systems wereexamined. An estimated percent consolidation of the lung of each pig wascalculated based on the scoring system described in Experiment XIIabove, in which each lung lobe was assigned a number to reflect theapproximate volume of entire lung represented by that lobe. Otherlesions were noted accordingly.

Microscopic Pathology:

Sections were taken from all lung lobes described above, as well as fromnasal turbinates, cerebrum, thalamus, hypothalamus, pituitary gland,brain stem, choroid plexus, cerebellum, heart, pancreas, ileum, tonsil,mediastinal lymph node, middle iliac lymph node, mesenteric lymph node,thymus, liver, kidney, and adrenal gland for histopathologicexamination. Tissues were fixed in 10% neutral buffered formalin for 1-7days and routinely processed to paraffin blocks in an automated tissueprocessor. Sections were cut at 6 μm and stained with hematoxylin andeosin. Lesions in several tissues were graded in accordance with thefollowing scale: (−)=normal, (+)=mild, (++)=moderate, (+++)=severe, and(++++)=very severe (see Table 19).

Results

Clinical Disease—Part (A), CDCD Pig Model:

VR 2385 challenged pigs demonstrated the most severe clinicalrespiratory disease, with scores above 2.5/6.0 on 7-9 DPI (Table 16).The onset of respiratory disease was noted on 3 DPI, and symptoms andlesions continued through 14 DPI. Respiratory disease was characterizedby labored and accentuated abdominal respirations and tachypnea. Therewas no coughing. The pigs became lethargic by 3 DPI, were anorexic by 5DPI, and did not return to full feed and activity until after 14 DPI.Eyelid edema was noted in two pigs on 6 and 7 DPI.

VR 2428-challenged pigs had a later onset of respiratory disease (5DPI), but severe respiratory disease occurred more quickly and for alonger duration than in ISU-12-inoculated pigs. VR 2428 producedrespiratory. scores greater than 3.0/6.0 on 7-13 DPI. The pigs were offfeed and lethargic at 6-14 DPI. No other clinical signs were noted.

ISU-984-challenged pigs produced moderate-to-severe respiratory diseasewith gradual onset starting at 4 DPI. The pigs were scored 2-2.5/6.0 forrespiratory disease from 7-10 DPI, and greater than 3.0/6.0 with a fewscores of 4-5/6.0 on 11-14 DPI. Other clinical signs included lethargy,eyelid edema, and blotchy-purple transient discoloration of skin.

VR 2431-challenged pigs produced mild respiratory disease. Disease onsetoccurred at 5 DPI with the most severe respiratory clinical diseasescores between 2 and 2.5/6.0 in some pigs at 7-8 DPI. The pigs appearedconsiderably better by 10 DPI and were completely normal by 14 DPI.Lethargy and anorexia were observed on 7-8 DPI.

Mean rectal temperatures were greater than 104° F. for all challengedgroups by 7 DPI, and remained above 104° F. until after 10 DPI. Thiscoincided with the period of most severe clinical respiratory disease.The control pigs remained clinically normal throughout the experiment.

Clinical Disease—Part (B), Comparative Pathogenicity:

Clinical respiratory disease scores and rectal temperatures aresummarized in Table 17. VR 2429 produced very mild respiratory diseaseand the pigs appeared near normal through 10 DPI. VR 2430 induced milddyspnea and tachypnea from 4-10 DPI, as well as lethargy and anorexiafrom 4-6 DPI. At 5-8 DPI, ISU-1894 produced moderate respiratory diseaseof short duration, and the pigs were generally recovered by 10 DPI.ISU-1894-inoculated pigs were also transiently lethargic and anorexicfrom 4-7 DPI. ISU-79 induced severe respiratory disease with laboredrespirations of increased frequency, accompanied by lethargy andanorexia from 4 DPI to 15 DPI. ISU-12 induced moderate tachypnea anddyspnea of long duration (4-28 DPI). These pigs were also moderatelylethargic and mildly anorexic over that time period.

Pigs in three groups (ISU-12, ISU-79, ISU-28) frequently exhibitedtransient, blue-purple discoloration of the skin when stressed byhandling. ISU-28 produced severe respiratory disease similar to ISU-79,but had a later onset (at 7 DPI) and only a 5-day duration. Controlsremained normal through 10 DPI.

Gross Lesions—Part (A), CDCD Pig Model:

Gross lung lesions were scored and estimated as percent lungconsolidation. Results are summarized in Table 16. The degree ofconsolidation ranged from 7.3% (ISU-984) to 29% (VR 2386) at 3 DPI, 20%(VR 2431) to 56.3% (VR 2386) at 7 DPI, 10.5% (VR 2431) to 77.5% (VR2385) at 10 DPI, 0% (VR 2431) to 37.3% at 21 DPI, and 0% (VR 2431, VR2385) to 11% (VR 2428) at 28 DPI. No grossly detectable lesions remainedin any group at 36 DPI. No gross lung lesions were observed at any timein the control group.

The affected lung lobes were primarily in the anterior, middle,accessory, and ventromedial portion of the caudal lobes. Theconsolidated areas were not well demarcated. These areas were multifocalwithin in each lobe and had irregular and indistinct borders, giving theaffected lobes a tan-mottled appearance. TABLE 16 Part (A) Mean GrossLung Consolidation 3 DPI 7 DPI 10 DPI 21 DPI 28 DPI Clin. Gross Clin.Gross Clin. Gross Clin. Gross Clin. Gross Isolate Score Lung Score LungScore Lung Score Lung Score Lung VR 2386 0.5 29 3.1 56.3 3.5 77.3 2.037.3 0.5 6.0 VR 2385 0.5 20.5 2.3 35.5 2.0 77.5 0.5 25.0 0 0.0 VR 2428 026.5 2.4 35.0 3.5 64.8 2.0 36.5 2.5 11.0 ISU-984 0.5 7.3 2.3 21.8 3.576.0 2.0 21.0 0 0.5 VR 2431 0 13.5 2.3 20.0 1.5 10.5 0 0 0 0.0 PSP-36 00 0 0 0 0 0 0 0 0.0 Uninoc. 0 0 0 0 0 0 0 0 0 0.0Gross Lesions—Part (B), Comparative Pathogenicity:

Gross lung lesions were estimated by percent lung consolidation, and areshown in Table 18.

Microscopic Lesions—Part (A), CDCD Pig Model:

Results are shown in Table 19. VR 2385, VR 2386, VR 2428 and ISU-984 allinduced similar microscopic lung lesions. They produced moderate-severeinterstitial pneumonia,-characterized by: (i) type II pneumocyteproliferation, (ii) septal thickening with mononuclear cells, and (iii)accumulation of mixed alveolar exudate. VR 2431 induced only mildinterstitial pneumonia with septal thickening by mononuclear cells.Myocarditis was observed only in the VR 2386 inoculated pigs.

Virus Isolation—Part (A), CDCD Pig Model:

PRRSV was recovered from the lungs of all 11 pigs inoculated with VR2386, from 9 of 11 pigs inoculated with VR 2385, from 6 of 11 pigsinoculated with ISU-984, from 9 of 11 pigs inoculated with VR 2431, from0 of 11 pigs inoculated with cell culture controls, and from 0 of 11uninoculated control pigs up to 28 DPI.

Serology—Part (A), CDCD Pig Model:

All of the PRRSV inoculated pigs had detectable PRRSV antibody titer of≧640 by 10 DPI. None of the control pigs had detectable PRRSV antibody.Most of the PRRSV-inoculated pigs had titers of ≧2560 by 28 DPI.

Serology—Part (B), Comparative Pathogenicity:

All of the PRRSV-inoculated pigs had PRRSV antibody titers of ≧64 by 10DPI. Control pigs did not have detectable PRRSV antibody.

Discussion

The 5-week-old CDCD pigs inoculated intranasally with 10^(5.8) TCID₅₀ ofPRRSV provide an excellent model to study and compare PRRSV-inducedrespiratory and systemic disease. Significant differences (p<0.05) wereobserved in the pneumopathogenicity data reported in Table 18. Based onthe results herein and in Experiment XI above, the isolates could begrouped into high and low virulence groups as follows:

-   -   high virulence: VR 2385, VR 2386, VR 2429 (ISU-22), ISU-28,        ISU-984, ISU-79    -   low virulence: VR 2431, VR 2428 (ISU-51), VR 2430, ISU-1894, LV

A PRRSV isolate may be considered to be a “high virulence” phenotype ifit results in one or more of the following:

-   -   (a) a mean gross lung consolidation at 10 DPI of at least 30%,        and preferably, at least 40%;    -   (b) moderate-to-very severe type II pneumocyte hypertrophy and        hyperplasia, moderate-to-very severe interstitial thickening,        moderate-to-very severe alveolar exudate, and the presence of        syncytia; or    -   (c) a mean respiratory distress score of at least 2.0 at some        point in time from 10-21 DPI.

Where an isolate does not meet any of the above criteria, it may beconsidered a “low virulence” phenotype.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein. TABLE 17 Part (B) Mean Respiratory Distress Scores and MeanRectal Temperature (° F.) Mean Respiratory Distress Score Mean RectalTemperature 3 5 7 10 15 21 28 3 5 7 10 15 21 28 Isolate DPI DPI DPI DPIDPI DPI DPI DPI DPI DPI DPI DPI DPI DPI PSP-36 0 0 0 0 0 0 0 102.7 102.6103.3 103.7 103.1 103.5 103.8 VR 2429 0 0.1 0.7 0.2 0 0.2 0 102.6 103.7104.2 103.2 104.5 103.6 104.2 VR 2430 0 1.1 0.8 1.5 0 0 0 102.8 103.7104.1 103.8 103.5 104.6 104.1 ISU-1894 0 2.5 1.5 1.1 0.5 0 0 102.7 104.4104.3 103.3 103.9 104.4 103.9 ISU-79 0 3.5 3.8 2.9 1.5 0.5 1.0 103.6104.9 104.6 103.7 103.4 103.5 103.8 VR 2385 0.2 1.5 1.4 1.4 1.0 2.4 0.8102.2 104.3 103.9 103.5 103.7 104.2 103.8 ISU-28 0 1.0 1.3 3.1 0 0 0102.6 104.2 104.0 104.8 104.0 103.8 103.9

TABLE 18 Part (B), Mean Gross Lung Consolidation and Standard DeviationNumber of Mean gross lung Inocula Pigs score 10 DPI SD PSP-36 10 0.0 0.0ISU-28 10 62.4 20.9 VR 2385 10 54.3 9.8 ISU-79 10 51.9 13.5 ISU-1894 1027.4 11.7 VR 2430 10 20.8 15.1 VR 2429 10 16.7 9.0

TABLE 19 Experiment XIII, part (A), CDCD pig model: Microscopic LesionSummary at 10 DPI VR VR VR VR PSP-36 Lesion 2386 2385 2428 ISU-984 2431control Type II pneumocyte ++++ +++ +++ +++ + − proliferation Syncytia++ ++ ++ ++ − − Interstitial ++++ +++ +++ +++ + − thickening alveolarexudate +++ +++ +++ +++ + − myocarditis + − − − − − encephalitis + − − −− −

1. A purified preparation containing a polynucleotide encoding at leastone polypeptide encoded by one or more open reading frames (ORF's) ofORFs 1-7 of a porcine reproductive and respiratory syndrome virus(PRRSV) wherein said polynucleotide has a sequence selected from thegroup consisting of the formulas (I), (II) and (III):5′-α-β-γ-3′  (I)5′-γ-δ-ε-3′  (II)5′-α-β-γ-δ-ε-3′  (III) wherein: α encodes at least one polypeptideencoded by a polynucleotide selected from the group consisting of ORF 1aand 1b, ORF 2 and ORF 3 of the PRRSV; β is either a covalent bond or alinking polynucleic acid which does not cause a decrease in the severityof gross and microscopic pneumonia lesions caused by the PRRSV; γ is atleast one copy of an ORF 5 from the PRRSV; δ is a covalent bond or alinking polynucleic acid which does not materially affect transcriptionand/or translation of said polynucleic acid; and ε encodes at least onepolypeptide encoded by a polynucleotide selected from the groupconsisting of ORF 6 and ORF 7 of the PRRSV; and when δ is a covalentbond, γ may have a 3′-end which excludes the region overlapping with the5′-end of a corresponding ORF
 6. 2. (canceled)
 3. A purified preparationcontaining a polynucleotide encoding at least one polypeptide encoded byone or more open reading frames (ORF's) of ORFs 1-7 of a porcinereproductive and respiratory syndrome virus (PRRSV) wherein the virus ischaracterized as highly virulent as determined by its ability to inducelesions in at least 51.9% of lung tissue 10 days post-inoculation offive-week-old colostrum-deprived, caesarean-derived pigs with 10⁵ TCID₅₀of said virus, and wherein said polynucleotide comprises multiple copiesof ORF
 5. 4.-5. (canceled)
 6. The purified preparation of claim 1,wherein said polynucleotide comprises ORF 2, ORF 3, ORF 5, ORF 6 or ORF7 of any one of VR 2385, VR 2386, VR 2429, VR 2484 or VR 2475, orcombinations thereof.
 7. The purified preparation of claim 1, whereinsaid polypeptide is encoded by at least one of ORF's 2, 3, 5, and 6 ofthe viruses VR 2385, VR 2386, VR 2429, VR 2484 or VR
 2475. 8-38.(canceled)
 39. A purified preparation containing a polynucleotideencoding at least one polypeptide encoded by one or more open readingframes (ORF's) of the viruses ISU-51 (VR 2428), ISU-55 (VR 2430),ISU-3927 (VR 2431) or ISU-1894 (VR 2475), wherein said polynucleotidehas a sequence selected from the group consisting of the formulas (I),(II) and (III):5′-α-β-γ-3′  (I)5′-γ-δ-ε-3′  (II)5′-α-β-γ-δ-ε-3′  (III) wherein: α encodes at least one polypeptideencoded by a polynucleotide selected from the group consisting of ORF 1aand 1b, ORF 2 and ORF 3 of the PRRSV; β is either a covalent bond or alinking polynucleic acid which does not cause a decrease in the severityof gross and microscopic pneumonia lesions caused by the PRRSV; γ is atleast one copy of an ORF 5 from the PRRSV; δ is either a covalent bondor a linking polynucleic acid which does not materially affecttranscription and/or translation of said polynucleic acid; and ε encodesat least one polypeptide encoded by a polynucleotide selected from thegroup consisting of ORF 6 and ORF 7 of the PRRSV; and when δ is acovalent bond, γ may have a 3′-end which excludes the region overlappingwith the 5′-end of a corresponding ORF
 6. 40. A purified preparationcontaining a polynucleotide encoding at least one polypeptide encoded byone or more open reading frames (ORF's) of the viruses ISU-51 (VR 2428),ISU-55 (VR 2430), ISU-3927 (VR 2431) or ISU-1894 (VR 2475), wherein saidORF 5 is from a PRRSV comprising multiple copies of ORF
 5. 41. Thepurified preparation of claim 39, wherein said polynucleotide comprisesORF 2, ORF 3, ORF 5, ORF 6 or ORF 7 of any one of VR 2428, VR 2430, VR2431, or VR 2475, or ORF combinations thereof.
 42. The purifiedpreparation of claim 39, wherein said polypeptide is encoded by at leastone of ORF's 2, 3, 5, and 6 of VR 2428, VR 2430, VR 2431, or VR 2475.43. A purified preparation of claim 1 or claim 39, containing one ormore polynucleotide at least (a) 98% identical to ORF 6 of the virus, or(b) 98% identical to ORF 7 of the virus; wherein the identity isdetermined using the following parameters: (i) a cost to open a gap 5;(ii) a cost to lengthen a gap of 25; (iii) a minimum diagonal length of4; and (iv) a maximum diagonal offset of
 10. 44. The purifiedpreparation of claim 43, wherein said polynucleotide has a sequenceselected from the group consisting of the formulas (I), (II) and (III):5′-α-β-γ-3′  (I)5′-γ-δ-ε-3′  (II)5′-α-β-γ-δ-ε-3′  (III) wherein: α encodes at least one polypeptideencoded by a polynucleotide selected from the group consisting of ORF 1aand 1b, ORF 2 and ORF 3 of the viruses of claim 43; β is either acovalent bond or a linking polynucleic acid which does not cause adecrease in the severity of gross and microscopic pneumonia lesionscaused by the PRRSV of claim 43; γ is at least one copy of an ORF 5 fromthe PRRSV of claim 43; δ is either a covalent bond or a linkingpolynucleic acid which does not materially affect transcription and/ortranslation of said polynucleic acid; and ε encodes at least onepolypeptide encoded by a polynucleotide selected from the groupconsisting of ORF 6 and ORF 7 of the PRRSV of claim 43; and when δ is acovalent bond, γ may have a 3′-end which excludes the region overlappingwith the 5′-end of a corresponding ORF
 6. 45. The purified preparationof claim 44, wherein said ORF 5 is comprised of multiple copies of ORF5.
 46. The purified preparation of claim 3, wherein said polynucleotidecomprises ORF 2, ORF 3, ORF 5, ORF 6 or ORF 7 of any one of VR 2385, VR2386, VR 2429, VR 2484 or VR 2475, or combinations thereof.
 47. Thepurified preparation of claim 3, wherein said polypeptide is encoded byat least one of ORF's 2, 3, 5, and 6 of the viruses VR 2385, VR 2386, VR2429, VR 2484 or VR
 2475. 48. The purified preparation of claim 3,containing one or more polynucleotide at least (a) 98% identical to ORF6 of the virus, or (b) 98% identical to ORF 7 of the virus; wherein theidentity is determined using the following parameters: (i) a cost toopen a gap 5; (ii) a cost to lengthen a gap of 25; (iii) a minimumdiagonal length of 4; and (iv) a maximum diagonal offset of
 10. 49. Thepurified preparation of claim 48, wherein said polynucleotide has asequence selected from the group consisting of the formulas (I), (II)and (III):5′-α-β-γ-3′  (I)5′-γ-δ-ε-3′  (II)5′-Ε-β-γ-δ-ε-3′  (III) wherein: α encodes at least one polypeptideencoded by a polynucleotide selected from the group consisting of ORF 1aand 1b, ORF 2 and ORF 3 of the viruses of claim 48; β is either acovalent bond or a linking polynucleic acid which does not cause adecrease in the severity of gross and microscopic pneumonia lesionscaused by the PRRSV of claim 48; γ is at least one copy of an ORF 5 fromthe PRRSV of claim 48; δ is either a covalent bond or a linkingpolynucleic acid which does not materially affect transcription and/ortranslation of said polynucleic acid; and ε encodes at least onepolypeptide encoded by a polynucleotide selected from the groupconsisting of ORF 6 and ORF 7 of the PRRSV of claim 48; and when δ is acovalent bond, γ may have a 3′-end which excludes the region overlappingwith the 5′-end of a corresponding ORF
 6. 50. The purified preparationof claim 40, wherein said polynucleotide comprises ORF 2, ORF 3, ORF 5,ORF 6 or ORF 7 of any one of VR 2385, VR 2386, VR 2429, VR 2484 or VR2475, or combinations thereof.
 51. The purified preparation of claim 40,wherein said polypeptide is encoded by at least one of ORF's 2, 3, 5,and 6 of the viruses VR 2385, VR 2386, VR 2429, VR 2484 or VR
 2475. 52.A purified preparation of claim 40, containing one or morepolynucleotide at least (a) 98% identical to ORF 6 of the virus, or (b)98% identical to ORF 7 of the virus; wherein the identity is determinedusing the following parameters: (i) a cost to open a gap 5; (ii) a costto lengthen a gap of 25; (iii) a minimum diagonal length of 4; and (iv)a maximum diagonal offset of
 10. 53. The purified preparation of claim52, wherein said polynucleotide has a sequence selected from the groupconsisting of the formulas (I), (II) and (III):5′-α-β-γ-3′  (I)5′-γ-δ-ε-3′  (II)5′-α-β-γ-δ-ε-3′  (III) wherein: α encodes at least one polypeptideencoded by a polynucleotide selected from the group consisting of ORF 1aand 1b, ORF 2 and ORF 3 of the viruses of claim 52; β is either acovalent bond or a linking polynucleic acid which does not cause adecrease in the severity of gross and microscopic pneumonia lesionscaused by the PRRSV of claim 52; γ is at least one copy of an ORF 5 fromthe PRRSV of claim 52; δ is either a covalent bond or a linkingpolynucleic acid which does not materially affect transcription and/ortranslation of said polynucleic acid; and ε encodes at least onepolypeptide encoded by a polynucleotide selected from the groupconsisting of ORF 6 and ORF 7 of the PRRSV of claim 52; and when δ is acovalent bond, γ may have a 3′-end which excludes the region overlappingwith the 5′-end of a corresponding ORF 6.